High throughput nucleic acid profiling of single cells
阅读说明：本技术 单细胞的高通量核酸谱分析 (High throughput nucleic acid profiling of single cells ) 是由 P·A·罗梅罗 L·B·海曼 于 2019-03-06 设计创作，主要内容包括：对单细胞的核酸组合物进行谱分析的方法和用于所述方法的工具。所述方法可以包含在液滴中分离单细胞,在所述液滴中裂解所述单细胞以从所述细胞释放模板核酸,在所述液滴中扩增所述模板核酸以产生经过扩增的核酸,以及在所述液滴中检测所述经过扩增的核酸。所述方法可用于对表达模式进行谱分析和/或检测如单核苷酸多态性等遗传特征。所述工具包含核酸逻辑门,包含聚合酶依赖性逻辑门。所述逻辑门可以执行如是、非、与、或、与非、非与、非或、异或、同或和蕴涵等逻辑运算。所述工具还包含用于执行所述方法的微流体系统。(Methods of profiling nucleic acid compositions of single cells and tools for use in the methods. The method may comprise isolating a single cell in a droplet, lysing the single cell in the droplet to release template nucleic acid from the cell, amplifying the template nucleic acid in the droplet to produce amplified nucleic acid, and detecting the amplified nucleic acid in the droplet. The methods can be used to profile expression patterns and/or to detect genetic characteristics such as single nucleotide polymorphisms. The tool comprises a nucleic acid logic gate comprising a polymerase dependent logic gate. The logic gates may perform logical operations such as, for example, yes, no, and, or, nand, not, nor, xor, xnor, and implication. The tool also includes a microfluidic system for performing the method.)
1. A method of profiling a nucleic acid composition of a single cell, the method comprising:
separating the single cell in a droplet;
lysing the single cells in the droplets to release template nucleic acids from the cells;
amplifying the template nucleic acid in the droplet to produce amplified nucleic acid; and
detecting the amplified nucleic acid in the droplet.
2. The method of claim 1, wherein the separating comprises separating the cells in aqueous droplets suspended in a water-immiscible medium.
3. The method of any one of claims 1-2, wherein said isolating comprises isolating said cells in said droplets with one or more of a lysis reagent, a DNA polymerase, an amplification primer, a deoxynucleotide triphosphate, an RNAse inhibitor, a nucleic acid logic gate, and a reporter.
4. The method of claim 3, wherein the lysis reagent comprises a detergent.
5. The method of claim 4, wherein the detergent comprises a non-ionic detergent.
6. The method according to any one of claims 3 to 5, wherein the DNA polymerase has DNA-dependent polymerase and/or RNA-dependent polymerase activity, has strand displacement activity, and lacks 5'→ 3' exonuclease activity.
7. The method of any one of claims 3-6, wherein the logic gate comprises a polymerase dependent logic gate.
8. The method of any one of claims 3-7, wherein the reporter comprises a non-specific fluorescent DNA reporter.
9. The method of any one of claims 3-7, wherein the reporter comprises a sequence-specific fluorescent DNA reporter.
10. The method of any one of claims 1-9, wherein the lysing comprises heating the cells in the presence of a lysing agent in the droplets.
11. The method of any one of claims 1-10, wherein the template nucleic acid comprises RNA and the amplifying comprises reverse transcribing the RNA to DNA.
12. The method of claim 11, wherein the amplifying further comprises amplifying the DNA.
13. The method of any one of claims 1-10, wherein the template nucleic acid comprises DNA and the amplifying comprises amplifying the DNA.
14. The method of any one of claims 1-13, wherein the amplifying comprises isothermally amplifying the template nucleic acid.
15. The method of any one of claims 1-14, wherein the amplifying comprises isothermally amplifying the template nucleic acid using enzyme-dependent isothermal amplification.
16. The method of any one of claims 1-15, wherein the amplification comprises loop-mediated isothermal amplification.
17. The method of any one of claims 1-16, wherein the detecting comprises non-specifically detecting the amplified nucleic acids.
18. The method of any one of claims 1-16, wherein the detecting comprises specifically detecting the amplified nucleic acid.
19. The method of any one of claims 1 to 18, wherein the detecting comprises performing a profiling analysis of the amplified nucleic acids by performing a molecular calculation with a nucleic acid logic gate.
20. The method of claim 19, wherein the logic gate is configured to perform a logical operation selected from the group consisting of: is, not, and, or, nand, not, xor, xnor, and inclusion.
21. The method of any one of claims 19-20, wherein the logic gate comprises a polymerase dependent nucleic acid logic gate.
22. The method of claim 21, wherein the polymerase dependent nucleic acid logic gate comprises an output strand annealed to a gate strand, the logic gate configured to release the output strand from the gate strand in the presence of an input strand and a DNA polymerase, wherein:
an output strand annealing recombination section that is recombined with the output strand annealing recombination section of the gate strand without annealing the output strand non-annealing recombination section that is recombined with the gate strand, wherein the output strand annealing recombination section is closer to the 5' end of the gate strand than the output strand non-annealing recombination section;
the input strand anneals to an input strand annealing rebinding moiety of the gate strand, and does not anneal to an input strand non-annealing rebinding moiety of the gate strand, wherein the input strand annealing rebinding moiety is closer to the 3' end of the gate strand than the input strand non-annealing rebinding moiety; and is
Annealing the input strand to re-bind to the gate strand and mediated extension of the input strand along the gate strand polymerase to form an extended input strand is necessary and sufficient to release the output strand from the gate strand.
23. The method of claim 22, wherein the output strand annealing rebinding moiety and the input strand non-annealing rebinding moiety at least partially overlap, and wherein the input strand annealing rebinding moiety and the output strand non-annealing rebinding moiety at least partially overlap.
24. The method of any one of claims 22 to 23, wherein the output strand annealing recombination section and the input strand annealing recombination section at least partially overlap.
25. The method of any one of claims 22 to 23, wherein the output strand annealing recombination events and the input strand annealing recombination events do not overlap.
26. The method of any one of claims 22 to 25, wherein the logic gate performs a not operation, wherein:
the logic gate further comprises a non-chain annealed to the output chain; and is
The release of the output chain from the gate chain causes the output chain to bind to the non-chain, thereby preventing downstream signaling or detection of the output chain.
27. The method of claim 26, wherein:
the output strand comprises a first of a fluorophore or a quencher of a fluorophore-quencher pair;
the non-strand comprises the fluorophore or the second of the quenchers of the fluorophore-quencher pair; and is
The release of the export strand from the gate strand results in binding of the export strand to the non-strand and quenching of the fluorophore.
28. The method of any one of claims 22-25, wherein the logic gate performs an exclusive-or operation, wherein:
the input chain comprises at least two different input chains;
the input strand annealing recombination portion of the gate strand is capable of annealing recombination to each of the at least two different input strands; and is
Annealing of any of the at least two different input strands to the gate strand induces polymerase-mediated extension of any of the at least two different input strands and displaces the output strand from the gate strand.
29. The method of any one of claims 22-25, wherein the logic gate performs an exclusive-or operation, wherein:
the input chain comprises at least two different input chains;
the door chain comprises at least two different door chains;
said input strand annealing recombination moiety of a first of said at least two different gate strands anneals to recombine to a first of said at least two different input strands; and is
The input strand annealing recombination moiety of a second of the at least two different gate strands anneals to recombine to a second of the at least two different input strands.
30. The method of any one of claims 22-25, wherein the logic gate performs an and operation, wherein:
the input chain comprises at least two different input chains;
the door chain comprises at least two different door chains;
said input strand annealing recombination moiety of a first of said at least two different gate strands anneals to recombine to a first of said at least two different input strands but does not anneal to recombine to a second of said at least two different input strands;
said input strand annealing recombination moiety of a second of said at least two different gate strands anneals to recombine with said second of said at least two different input strands but does not anneal to recombine with said first of said at least two different input strands;
the logic gate further comprises a threshold chain, wherein the threshold chain is present in the droplet at a substantially equal molar concentration as each of the at least two different gate chains to which the output chain binds, and wherein the threshold chain anneals to re-bind to the output chain when displaced from the gate chains; and is
The first of the at least two different gate chains anneals to its input chain annealing recombination moiety and the second of the at least two different gate chains anneals to its input chain annealing recombination moiety are necessary to release an amount of output chains that exceed the threshold chain.
31. The method of any one of claims 22-25, wherein the logic gate performs a nand operation, wherein:
the input chain comprises at least two different input chains;
the door chain comprises at least two different door chains;
the output strands include at least two different, substantially complementary output strands;
said input strand annealing recombination moiety of a first of said at least two different gate strands anneals to recombine to a first of said at least two different input strands but does not anneal to recombine to a second of said at least two different input strands;
said input strand annealing recombination moiety of a second of said at least two different gate strands anneals to recombine with said second of said at least two different input strands but does not anneal to recombine with said first of said at least two different input strands; and is
The at least two different output strands anneal to recombine with each other when released from the gate strand.
32. The method of any one of claims 22 to 25, wherein:
the output strand includes a reporter gate annealing recombination moiety that anneals to the reporter or quencher strand of the reporter gate.
33. The method of claim 32, wherein:
the output strand further comprises a second input strand annealing recombination moiety that anneals to a second input strand, wherein the second input strand annealing recombination moiety is closer to the 3' end of the output strand than the reporter-gated annealing recombination moiety.
34. The method of any of claims 22-25, wherein the logic gate further comprises a threshold chain configured to anneal to recombine to the output chain.
35. The method of any one of claims 22-34, wherein the gate strand is about 4 to about 300 nucleotide bases in length.
36. The method of any one of claims 22-35, wherein the output strand is about 2 to about 100 nucleotide bases in length.
37. The method of any one of claims 22-36, wherein the input strand comprises the amplified nucleic acid.
38. The method of any one of claims 22 to 36, wherein the input chain is an output chain from a second logic gate configured to detect a second input chain.
39. The method of any one of claims 22 to 38, wherein: the output chain is an input chain of second logic gates.
40. The method of any one of claims 22 to 39, wherein the output chain is an input chain of reporter gates.
41. The method of any one of claims 19-40, wherein the lysing, the amplifying, and the profiling the amplified nucleic acids are performed at substantially the same temperature.
42. The method of any one of claims 19 to 41, wherein said lysing, said amplifying, and said profiling said amplified nucleic acids are performed at a substantially constant temperature.
43. The method of any one of claims 19 to 42, wherein a substantially constant temperature is maintained throughout and between the lysing, the amplifying, and the profiling of the amplified nucleic acids.
44. The method of any one of claims 1-7, 10-16, and 19-43, wherein the detecting comprises detecting the amplified nucleic acids with a non-specific fluorescent DNA reporter.
45. The method of any one of claims 1-7, 10-16, and 19-43, wherein the detecting comprises detecting the amplified nucleic acid with a specific fluorescent DNA reporter.
46. The method of any one of claims 1-45, wherein the detecting comprises detecting a single nucleotide polymorphism in the nucleic acid composition of the single cell.
47. The method of any one of claims 1-46, wherein said separating, said lysing, said amplifying, and said detecting all occur in a single, continuous network of channels.
48. The method of any one of claims 1-47, wherein said separating occurs prior to said lysing, said lysing occurs prior to said amplifying, and/or said amplifying occurs prior to said detecting.
49. The method of any one of claims 1 to 48, wherein said droplets maintain a substantially constant volume throughout and between said lysing, said amplifying, and said detecting.
50. The method of any one of claims 1 to 49, wherein the lysing, the amplifying, and the detecting all occur without diluting the droplets, adding additional reagents to the droplets, and/or removing reagents or liquid from the droplets after the separating.
51. A polymerase dependent nucleic acid logic gate according to any one of claims 21 to 40.
52. A system for performing the method of any of claims 1-50.
Technical Field
The present invention relates to high throughput methods, systems, and devices for profiling nucleic acid compositions of single cells in a heterogeneous population of cells.
Background
Cellular heterogeneity and its impact on biological function and disease are becoming increasingly important in human immunology, stem cell biology and cancer research. For example, by performing transcription analysis on individual cells using reverse transcriptase polymerase chain reaction (RT-PCR), it is possible to identify rare cells or transient cell states that cannot be observed when studying the entire population in large numbers (Bendall et al, 2012; Kalisky and Blainey et al, 2011; Kalisky and Quake, 2011; Levsky et al, 2003). However, obtaining meaningful information about these cells requires tools that enable high throughput analysis. At present, the method for performing spectrum analysis on single cells by using RT-PCR operation, separation and transcription is complex, the yield is limited, and only hundreds of single cells can be detected.
The ultra-high throughput capability of droplet-based microfluidics is an ideal choice for single cell analysis applications (Guo et al, 2012; Novak et al, 2011; Vyawahare et al, 2010). These microfluidic technologies rely on microdroplets (tiny aqueous liquid spheres of 1 to 100 μm in diameter) to encapsulate biological components in oil-based emulsions (The et al, 2008). These droplets essentially act as very small "cuvettes" separating millions of reactions. One of the main advantages of this method is that a minimum amount of reagents is used, thereby greatly reducing the cost of a given experiment. Furthermore, using microfluidic technology, droplets may be formed and separated, reagents injected into them, and sorted at kilohertz rates, making it possible to perform millions of single-cell reactions at previously unavailable throughput. However, one obstacle to achieving the potential of this approach is that at the concentration of single cells in the microdroplets, the cell lysate is a potent inhibitor of RT-PCR (Arezi et al, 2010; Hedman et al, 2013; White et al, 2011).
To avoid inhibition of RT-PCR by cell lysates, previous droplet-based methods have used larger droplets (2nL) where the concentration of lysate is no longer inhibitory, or used agarose droplets that can be coagulated, rinsed and stained with DNA dyes (Mary et al, 2011; Zhang et al, 2012). Methods using large droplets can only analyze a total of about 100 cells, whereas agarose methods cannot use TaqMan probes or cell staining, thereby precluding the association of specific cell types with relevant transcriptional targets. Another strategy for single cell RT-PCR of cells is to isolate the cells into microwells fabricated as flexible devices. This method allows for robust and specific single cell transcriptional profiling. However, since each microwell and its control valves must be fabricated and individually controlled, the flux is also limited to only a few hundred total cells (Kalisky and Blainey et al, 2011; Kalisky and Quake, 2011; White et al, 2013). Another strategy for single cell RT-PCR on single cells is to lyse the cells in a microdroplet and then dilute the microdroplet before RT-PCR (Eastburn et al, 2013). While this dilution method is effective, it is a very cumbersome process that limits processability.
To be able to perform expression analysis on a large number of cells in a heterogeneous population, new methods, systems and devices that combine the throughput of droplet-based microfluidics with the specificity of microwell reactions are needed. Aspects of the invention provided herein address these needs.
Disclosure of Invention
One aspect of the invention relates to a method of profiling a nucleic acid composition of a single cell. The method includes isolating a single cell in a droplet, lysing the single cell in the droplet to release a template nucleic acid from the cell, amplifying the template nucleic acid in the droplet to produce an amplified nucleic acid, and detecting the amplified nucleic acid in the droplet.
Another aspect of the invention relates to a polymerase dependent logic gate. The polymerase dependent logic gate can be used in the profiling methods of the present invention.
Another aspect of the invention relates to a microfluidic system for performing the spectral analysis method of the invention.
The objects and advantages of the present invention will become more fully apparent from the following detailed description of the preferred embodiments of the invention, taken in conjunction with the accompanying drawings.
Drawings
FIG. 1 shows a schematic diagram of an exemplary form of the process of the present invention.
FIG. 2 shows a schematic diagram of an exemplary branch migration-mediated strand displacement reporter. The half arrow indicates the 3' end of the depicted nucleic acid strand. In fig. 2-4, the nucleic acid portion represented by a given number (e.g., "1") is substantially identical to the nucleic acid portion represented by the same number and is substantially complementary to the nucleic acid portion represented by the same number and asterisk (e.g., "1").
FIG. 3 illustrates an exemplary branch migration mediated strand permutation logic gate that employs logical operations. The half arrow indicates the 3' end of the depicted nucleic acid strand.
FIG. 4 shows a schematic diagram of an exemplary polymerase dependent logic gate employing a logical operation. The half arrow indicates the 3' end of the depicted nucleic acid strand.
FIG. 5 shows a schematic diagram of an exemplary polymerase dependent logic gate employing an OR logic operation. The half arrow indicates the 3' end of the depicted nucleic acid strand.
FIG. 6 shows a schematic diagram of an exemplary polymerase dependent logic gate employing an AND logic operation. The half arrow indicates the 3' end of the depicted nucleic acid strand.
FIGS. 7A-7D illustrate schematic diagrams of alternative exemplary polymerase dependent logic gates employing an AND logic operation. Fig. 7A shows the components of the logic gate. Fig. 7B shows the operation of the logic gate in the case where both target inputs are present. Fig. 7C and 7D show the operation of the logic gate in the case where only one of the two target inputs is present. The half arrow indicates the 3' end of the depicted nucleic acid strand. The logic gates shown in fig. 7A may constitute an exclusive or logic operation in the absence of a threshold chain.
FIG. 8 shows a schematic diagram of an exemplary polymerase dependent logic gate employing a NAND logic operation. The half arrow indicates the 3' end of the depicted nucleic acid strand.
FIGS. 9A-9D illustrate schematic diagrams of alternative exemplary polymerase dependent logic gates employing NAND logic operations. The half arrow indicates the 3' end of the depicted nucleic acid strand. Fig. 9A shows the components of the logic gate. Fig. 9B shows the operation of the logic gate in the case where both target inputs are present, where the presence of
FIG. 10 shows a schematic diagram of an exemplary polymerase dependent logic gate employing a non-logical operation. The half arrow indicates the 3' end of the depicted nucleic acid strand.
Fig. 11 shows a schematic using an exemplary polymerase dependent logic gate that is a logical operation that uses the 3' end of the LAMP dumbbell product as an input. The half arrow indicates the 3' end of the depicted nucleic acid strand.
Fig. 12 illustrates an exemplary apparatus suitable for performing the methods described herein.
Fig. 13 illustrates an exemplary apparatus for sorting droplets performed in the methods described herein.
FIGS. 14A and 14B show the effect of lysate on Catalytic Hairpin Assembly (CHA) amplification. Figure 14A shows a high background using CHA in the presence of lysate. Figure 14B shows concentration dependent lysate inhibition. In FIG. 14A, error bars represent +/-1 standard deviation of the mean.
FIGS. 15A and 15B show RT-LAMP amplification of mRNA for the epithelial marker CK19(KRT19) in CK19+ human breast cancer cells (SK-BR-3) and CK 19-white blood cells (MOLT-4). FIG. 15A shows 106Amplification at a lysate concentration of one cell/ml, which corresponds to the lysate concentration of a single cell in the microdroplet. FIG. 15B shows above and below 106Amplification of individual cells/ml lysate concentration. Error bars represent +/-1 standard deviation of the mean.
Fig. 16 shows the fluorescence detection pattern of LAMP products using the epithelial phenotype marker KRT19 and/or the mesenchymal phenotype marker VIM with a polymerase dependent logic gate that is a logical, non-logical, or logical, and logical, or non-logical operation. It is the logic gate that is constructed as shown in fig. 4. The not logic gate is constructed as shown in fig. 10. The or logic gate is constructed as shown in fig. 7A-7D, with the threshold chain omitted. The and logic gate is constructed as shown in fig. 7A-7D. The nand logic gate is constructed as shown in fig. 9A-9D.
FIGS. 17A and 17B show fluorescence detection of KRT19 RNA (FIG. 17A) or VIM RNA (FIG. 17B) using RT-LAMP with multiplex primer format and constructed as shown in FIG. 4, which is a nucleic acid logic gate. Error bars represent +/-1 standard deviation of the mean.
Figures 18A and 18B show fluorescence detection of KRT19 and/or VIM RNA using RT-LAMP and or logic gates (figure 18A) or and logic gates (figure 18B). Both the or gates and the logic gates are constructed as shown in fig. 7A-7D, except that the or logic gates lack a chain of thresholds. Error bars represent +/-1 standard deviation of the mean.
Figures 19A and 19B show fluorescence detection of KRT19 and/or VIM RNA using RT-LAMP and a non-logic gate (figure 19A) or a non-logic gate (figure 19B). The not-gate is constructed as shown in fig. 10, and the nand-gate is constructed as shown in fig. 9A-9D. Error bars represent +/-1 standard deviation of the mean.
Figures 20A and 20B show RT-LAMP amplification of mRNA from the epithelial marker CK19(KRT19) in CK19+ human breast cancer cells (SK-BR-3) (figure 20A) and CK 19-white blood cells (MOLT-4) (figure 20B) in aqueous droplets suspended in fluorinated oil, using dsDNA specific dyes to indicate amplification.
Figure 21 shows a histogram of RT-LAMP amplification of Estrogen Receptor (ER) mRNA transcript ESR1 in ER + human breast cancer cells (MCF7) versus RT-LAMP amplification of ER-breast cancer cells (SK-BR-3) in aqueous droplets suspended in fluorinated oil, using dsDNA-specific dyes to indicate amplification. Prior to detection, droplets were collected and heated in batches.
FIG. 22 shows a histogram of RT-LAMP amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA transcripts in the human leukocyte cell line MOLT-4 in aqueous droplets suspended in fluorinated oil, using dsDNA-dependent dyes to indicate amplification. The droplets are generated, incubated and analyzed in an integrated microfluidic device that implements the entire workflow.
Fig. 23A and 23B show histograms of RT-LAMP amplification using multiplex and orthogonal epithelial marker CK19(KRT19) and mesenchymal marker VIM (VIM) of the logic gate and reporter complex, where KRT19 amplification activates AlexaFluor 647 reporter and VIM amplification activates HEX reporter. FIG. 23A shows histograms of Alexa Fluor 647 signals of human CK 19-osteosarcoma cell line (U-2OS) and human CK19+ breast cancer cell line (SK-BR-3) after 60 min or 5 min incubation. FIG. 23B shows histograms of HEX signals of human VIM-breast cancer cell line (SK-BR-3) and human VIM + osteosarcoma cell line (U-2OS) after 60 or 5 minutes incubation. Each is a gate configured as shown in fig. 4, and each reporter complex is configured as shown in fig. 2.
FIGS. 24A and 24B show droplets collected from the waste outlet (FIG. 24A) or the sort outlet (FIG. 24B) of a droplet sorting device that discriminates between the human ER + breast cancer cell line MCF7 and the ER-breast cancer cell line SK-BR-3 based on ESR1(ER) LAMP amplification.
Figure 25 shows LAMP-based SNP detection in total RNA samples from two cell lines: SK-BR-3, with WT ACTB transcript only (SEQ ID NO: 1); and MOLT-4, which contains the A- > C SNP at position 960 of the ACTB transcript (SEQ ID NO: 2). Error bars represent +/-1 standard deviation of the mean.
Detailed Description
One aspect of the invention relates to a method of profiling a nucleic acid composition of a single cell. The method may include isolating a single cell in a droplet, lysing the single cell in the droplet to release template nucleic acid from the cell, amplifying the template nucleic acid in the droplet to produce amplified nucleic acid, and detecting the amplified nucleic acid in the droplet.
Fig. 1 shows a schematic diagram of an exemplary form of this process. This exemplary procedure was used to profile nucleated blood cells for the presence of Circulating Tumor Cells (CTCs). The isolation in fig. 1 is described as "encapsulation of single cells", lysis as "cell lysis", amplification as "amplification", and detection as "DNA-based logical computation" and "high-throughput fluorescence detection". As shown in fig. 1, separation, lysis, amplification and detection can all occur in a continuous, high-throughput manner in a single network of channels. This is in contrast to processes where certain steps are performed in batches. The channel may be a microfluidic channel created using any suitable method. An exemplary method is provided in the following example. As used herein, "channel" refers to a channel embedded in a solid device as well as an exposed conduit.
The separating may comprise separating the cells in aqueous droplets suspended in a water immiscible medium. This may be performed by feeding the cells through the microfluidic channel in the form of a continuous stream of aqueous solution through an inlet of a water immiscible medium. The water-immiscible medium disrupts the continuous aqueous solution stream and "pinches off" the different droplets, causing them to become suspended in the water-immiscible medium. The cells may be present in the continuous stream of aqueous solution at a sufficiently low concentration that one or fewer cells are separated from each formed aqueous droplet. The water-immiscible medium may comprise an oil. An exemplary oil is a fluorinated oil, such as QX200 for EvaGreen #1864005TMThe droplets produced oil (BioRad, Hercules, CA) by burle corporation of herrales, california. Various surfactants present in the water-immiscible medium and/or the aqueous phase may help stabilize the droplets in the water-immiscible medium. The droplets preferably have a volume of from about 1pL to about 100nL, such as from about 10pL to about 10nL or from about 100pL to about 1 nL. Amounts above and below these amounts are acceptable.
For downstream steps such as lysis, amplification and detection, it is preferred to isolate each cell in the droplet with one or more reagents (e.g., lysis reagent, DNA polymerase, amplification primers, deoxynucleotide triphosphates, RNase inhibitor, one or more nucleic acid logic gates and a reporter). This may be performed by combining one or more solutions containing these reagents with a solution containing cells upstream of the droplet formation. The solution may form an adjacent laminar flow prior to droplet formation. The solution is co-encapsulated by a water-immiscible medium to form droplets. Droplet formation thus isolates individual cells with reagents.
The lysis reagent that is separated from the cells in the droplets may comprise a detergent. The detergent preferably comprises a non-denaturing detergent. The non-denaturing detergent preferably comprises a non-ionic detergent. Exemplary non-denaturing nonionic detergents include Tween 20 (Millipore Sigma, Burlington, MA), TRITON X-100 (Dow Chemical Company, Midland, MI, Midland, Mich.) 4- (1,1,3, 3-tetramethylbutyl) phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol t-octylphenyl ether), NP-40 (nonylphenoxypolyethoxyethanol), NONIDET P-40 (Shell Chemical Co., The Hague, The Netherlands) (octylphenoxypolyethoxyethanol), and The like. The detergent is preferably present in the droplets at a concentration of about 0.5% v/v to about 5% v/v, such as a concentration of about 1% v/v to about 5% v/v or a concentration of about 2.5% v/v. The cleavage reagent may alternatively or additionally comprise a cleaving enzyme. Exemplary lytic enzymes include lysozyme and phage lytic enzymes. Suitable lysozyme concentration of lysozyme comprises a concentration between about 1kU/ml and 60kU/ml, such as about 30 kU/ml. Concentrations above and below these amounts are also acceptable. The optimal concentration of other specific lytic enzymes can be readily determined. The detergent should be sufficient to lyse most cells, especially when heated. For cells having a cell wall, such as certain types of bacteria, the lytic reagent preferably comprises a detergent in addition to the lytic enzyme. In some forms of the invention, no lysing agent is present, and cell lysis occurs by heating alone.
The polymerase that is separated from the cells in the droplets can be any polymerase suitable for downstream amplification of the target nucleic acid. For amplification of a DNA target nucleic acid, the polymerase is preferably a DNA-dependent DNA polymerase. DNA-dependent DNA polymerases are enzymes that catalyze the replication of DNA in a DNA template. An exemplary DNA-dependent DNA polymerase is Taq polymerase. For amplification of RNA target nucleic acids, the polymerase is preferably a DNA-dependent and RNA-dependent DNA polymerase or a combination of a DNA-dependent DNA polymerase and a RNA-dependent DNA polymerase alone. RNA-dependent DNA polymerases are enzymes that catalyze the production of DNA from an RNA template. RNA-dependent DNA polymerases are sometimes also referred to as "reverse transcriptases". DNA-dependent and RNA-dependent DNA polymerases are enzymes having both DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase (reverse transcriptase) activity. "amplification" as used herein is the same as that generally used in the art, except that it is also understood herein to encompass reverse transcription of an RNA target nucleic acid into DNA.
For certain types of amplification (e.g., isothermal amplification), the polymerase additionally has strand displacement activity and lacks 5'→ 3' exonuclease activity. The polymerase may also contain or lack 3'→ 5' exonuclease activity. The polymerase is preferably thermostable. An exemplary polymerase is Bst 2.0DNA polymerase (New England BioLabs, inc., Ipswich, MA). This enzyme has RNA-dependent DNA polymerase (reverse transcriptase) activity, DNA-dependent DNA polymerase activity and strand displacement activity, and lacks 5'→ 3' exonuclease activity. Other polymerases having these characteristics are well known in the art.
Amplification primers isolated with cells in the droplets comprise any primers suitable for amplification of a nucleic acid template. Many amplification methods and the design of suitable primers therefor are known in the art. In amplification, the dNTPs serve as building blocks for the amplified nucleic acid.
The nucleic acid logic gates separated from the cells in the droplet may include any one or more nucleic acid logic gates suitable for profiling nucleic acids. Exemplary nucleic acid logic gates are known in the art and are described elsewhere herein.
The reporter isolated with the cells in the droplet can include any agent suitable for generally indicating the presence of a nucleic acid or specifically indicating the presence of a particular nucleic acid. Exemplary reporters are known in the art and are described elsewhere herein.
The lysing step may include heating the cells in the presence or absence of a lysing agent in the droplets. This step may be performed by heating the droplet containing the cells or the cells and the lysis reagent. Heating the droplets may be performed by flowing the droplets containing the cells or the cells and the lysis reagent through a heated zone. The heated region may comprise the entire device comprising the channel or a sub-portion thereof. In some forms, no heating step is required, as mixing the lysis reagent with the cells is sufficient to lyse the cells themselves.
The amplification may comprise amplifying the DNA template or reverse transcribing the template RNA into DNA, and amplifying the reverse transcribed DNA. The type of amplification used depends on the type of nucleic acid used for the profiling. For example, a DNA template typically requires only amplification of the DNA itself. In contrast, RNA templates typically require reverse transcription and DNA amplification.
The amplification method may comprise any method suitable for amplifying nucleic acids. Exemplary methods include PCR methods and isothermal amplification methods. Reverse transcription may be combined with PCR or isothermal amplification methods. Exemplary isothermal amplification methods include an enzyme-free isothermal amplification method and an enzyme-dependent isothermal amplification method. Exemplary enzyme-free isothermal amplification methods include Hybrid Chain Reaction (HCR), Catalytic Hairpin Assembly (CHA), and the like. Exemplary enzyme-dependent isothermal amplification methods include loop-mediated isothermal amplification (LAMP), Rolling Circle Amplification (RCA), Multiple Displacement Amplification (MDA), Recombinase Polymerase Amplification (RPA), nucleic acid sequence-based amplification (NASBA), and the like.
The presence of lysis reagents and/or undiluted cell lysate during amplification may inhibit certain amplification methods, such as PCR methods and enzyme-free isothermal amplification methods (e.g., HCR and CHA). As shown in the examples below, LAMP is capable of generating a strong amplification signal in the presence of lysis reagents and undiluted cell lysate. Thus, preferred amplification methods carried out in the presence of lysis reagents and/or undiluted cell lysate include enzyme-mediated isothermal amplification methods, such as LAMP.
Following nucleic acid amplification, the amplified nucleic acid can be detected by a variety of methods. Preferred methods include fluorescence. The method may comprise non-specific nucleic acid detection and specific nucleic acid detection.
Non-specific nucleic acid detection nucleic acids are detected using non-specific nucleic acid reporters (e.g., non-specific fluorescent DNA reporters) regardless of the specific sequence. Exemplary non-specific nucleic acid reporters include ethidium bromide, propidium iodide, crystal violet, dUTP conjugated probes, DAPI (4', 6-diamino-2-phenylindole), 7-AAD (7-aminomycin D), Hoechst 33258, Hoechst 33342, Hoechst 34580, picocreen (molecular probes, inc., Eugene, OR), Helixyte (AAT biological probes, AATBioquest, Sunnyvale, CA), YOYO-1, DiYO-1, TOTO-1, DiTO-1, and SYBR dyes (molecular probes, eugold, oregon, such as SYBR Green I, SYBR Green II, SYBR D, etc.). Many of these non-specific nucleic acid reporters are DNA intercalators.
Specific nucleic acid detection nucleic acid species having a specific sequence are detected using a sequence specific nucleic acid reporter (e.g., a sequence specific fluorescent nucleic acid reporter). Many sequence-specific nucleic acid fluorescent reporters are known in the art. Exemplary sequence-specific fluorescent nucleic acid reporters comprise a quenching reporter that releases a quencher upon binding to a particular sequence. An example of a sequence specific fluorescent nucleic acid reporter is shown in figure 2. This reporter is configured to operate through branch migration mediated strand displacement. The reporter includes a quencher strand 22 (black) that anneals to and re-binds to a substantially complementary reporter strand 20 (red). The quencher chain 22 includes a quencher 23 (Q). The reporter strand 20 includes a fluorophore 21 (F). Annealing and re-binding of the quencher strand 22 to the reporter strand 20 places the quencher 23 in proximity to the fluorophore 21, thereby quenching any fluorescence emitted by the fluorophore 21. The reporter strand 20 anneals only to the first portion (1) of the quencher strand 22, leaving the second portion (2) of the quencher strand 22 (called the sticky end) exposed. The quencher strand 22 is designed to be substantially complementary to the input strand 24 (blue). The input strand 24 is the nucleic acid to be detected. The input strand 24 includes a portion (2) that is substantially complementary to the sticky end (1). When present, the input strand 24 binds to the sticky end through a substantially complementary moiety and displaces the reporter strand 20 from the quencher strand 22 upon annealing to re-bind to the remainder of the quencher strand 22. Displacement of the reporter strand 20 removes the fluorophore 21 from the vicinity of the quencher 23, resulting in an activated reporter (shown as "output" in fig. 2). The activated reporter may fluoresce under optical excitation. In another form, the reporter strand is substantially complementary to the input strand and includes a sticky end, such that binding of the input strand to the sticky end on the reporter strand and subsequent branch migration displaces the quencher strand from the reporter strand. The resulting input strand/reporter strand duplex is capable of fluorescing.
Detection may include performing a spectroscopic analysis of the composition of amplified nucleic acids by performing molecular logic calculations using molecular logic loops. The molecular logic circuit inputs one or more specific species of nucleic acids, performs logical calculations, and outputs one or more different species of nucleic acids. Molecular logic circuits include one or more nucleic acid logic gates used alone or in various combinations. As used herein, "molecular logic computation" or "molecular computation" refers to the generation of one or more output nucleic acids (e.g., output strands) from one or more nucleic acid logic gates in response to one or more input nucleic acids (e.g., input strands). "generating" herein refers to the displacement of an output nucleic acid from a nucleic acid logic gate such that the output nucleic acid can be detected or used as an input to one or more downstream nucleic acid logic gates, as described in further detail below. The molecular logic loop may be a DNA-based molecular logic loop containing one or more DNA logic gates, an RNA-based molecular logic loop containing one or more logic gates, or a combination thereof.
Each logic gate is configured to perform a particular logical operation. Exemplary logical operations include yes, no, and, or, nand, not, nor, xor, xnor, and implication.
It is the gate and not gate that are each configured to perform spectral analysis on a single input.
It is the gate that produces output (1) if and only if the input abundance is high. This means that the gate cannot produce an output (0) if the input abundance is not high. A door may also be referred to as a "transducer". The logical operation performed by the yes gate on input a is shown in the following truth table:
a not gate is a loop that produces an inverted version of the input at its output. It is also called an inverter. If the input variable is A, the inverted output is not A. This is also shown as A', or A with a bar on top. The logical operation performed by the not gate on input a is shown in the following truth table:
and, or, nand, not, nor, xor, and xnor operations are each configured to perform spectral analysis on a plurality of inputs.
The and gate is configured to produce a high output (1) only when all its inputs are high, otherwise it cannot produce a high output (0). The AND operation is represented using a point (.): and (A) and (B). Points are sometimes omitted: and AB. The logical operations performed by the AND gates on inputs A and B are shown in the following truth table:
if one or more of the inputs is high, an OR gate (also referred to as a doubleOR gate) is configured to produce a high output (1). The or operation is represented using a plus sign (+). The logical operations performed by the OR gates on inputs A and B are shown in the following truth table:
a Not and (NAND) gate is equal to an and gate followed by a not gate. If either input is low (0), the outputs of all NAND gates are high (1). The logical operation performed by the NAND gate on inputs A and B is shown in the following truth table:
a nand gate detects the presence of only one of two possible inputs. If one of the two possible inputs and only one is high (1), the output of the nand gate is high (1), and if any other condition is obtained, the output is low (0). The truth table of the nand gate negating input B (a and B) is as follows:
a Not Or (NOR) gate is equal to an or gate followed by a not gate. If either input is high (1), the outputs of all NOR gates are low (0). The logical operations performed by the NOR gate on inputs A and B are shown in the following truth table:
an exclusive-or (EXOR) gate is configured to produce a high output if one, but not both, of its two inputs is high (1). The EXOR operation is represented using a circled plus sign. The logical operations performed by the exclusive or gates on inputs a and B are shown in the following truth table:
the exclusive nor (EXNOR) gate functions in opposition to the EXOR gate. The exclusive-nor gate is configured to produce a high output if both inputs are high (1) or both inputs are low (0). If one (but not both) of its two inputs is high, a low output is produced. The logical operations performed by the exclusive-nor gates on inputs a and B are shown in the following truth table:
the implication gate uses the CONDITIONAL logical operation by passing the 'if-then' logic.
Nucleic acid logic gates configured to perform logical operations are known in the art. See, e.g., Baccouche et al, 2014; chen et al, 2015; deng et al, 2014; li et al, 2011; li et al, 2013; li et al, 2016; massey et al, 2017; okamoto et al, 2004; qian, Winfree, 2011; qian, Winfree et al, 2011; ravan et al, 2017; thunage et al, 2017; wei et al, 2016; xu et al, 2014; yang et al, 2013; yang et al, 2016; yang et al, 2014; yao et al, 2015; zhang et al, 2010; zhu et al, 2013; zuo et al, 2017; US 2007/0072215 and WO 2017/141068.
Exemplary logic gates operate by branch migration mediated strand displacement, also referred to as sticky end-mediated branch migration or random walk branch migration. FIG. 3 illustrates a schematic diagram of a chain replacement logic gate with exemplary branch migration mediation that is a logical operation. The logic gates include an output strand 33 (brown strand) annealed to a substantially complementary gate strand 32 (blue strand). The input strand 31 (green), which is substantially complementary to the
Other exemplary logic gates include polymerase dependent logic gates. Polymerase dependent logic gates require polymerase mediated extension of the input strand along the gate strand to displace the output strand from the gate strand. These logic gates are designed to operate with a polymerase having strand displacement activity but lacking 5'→ 3' exonuclease activity. A schematic diagram of an exemplary polymerase dependent logic gate employing a logical operation is shown in fig. 4. The polymerase dependent logic gate includes annealing the export strand 43 (brown) which is re-bound to the gate strand 42 (blue). Output strand 43 is annealed and recombined at the output strand annealing and recombination portion (2) of gate strand 42. Output link 43 is not annealed and recombined with the output link non-annealed and recombined portion (1 ×) of gate link 42, leaving an exposed portion on gate link 42. The annealed recombined portion of the output strand is closer to the 5' end of the gate strand 42 (brown) than the non-annealed recombined portion of the output strand. Input strand 41 (green) anneals to the input strand annealing recombination section (1) of gate strand 42. In the example of fig. 4, the input strand annealing recombination section of the gate chain 42 is the same as the output strand non-annealing recombination section, but this is not necessarily so. Input strand 41 does not anneal to the input strand non-annealing recombination portion (2x) of gate strand 42. In the example of fig. 4, the input chain non-annealing recombination section of the gate chain 42 is the same as the output chain annealing recombination section, but this is not necessarily so. The input chain annealing recombination section that merely couples input chain 41 to gate chain 42 is not sufficient by itself to displace output chain 43 from gate chain 42. In order to displace output chain 43 from gate chain 42, extension of input chain 41 along gate chain 42 must occur. The output chain 43 may then be detected directly with a reporter gate, or the output chain may serve as an input to one or more further logic gates.
The input strand non-annealing recombination section of the gate strand does not necessarily have to be identical to the output strand annealing recombination section. However, in order to have an exposed portion near the 3' end of the input strand to facilitate the binding of the input strand, the input strand annealing rebinding portion and the output strand non-annealing rebinding portion at least partially overlap. In some forms, the input strand annealing rebinding moiety is a sub-moiety of the output strand non-annealing rebinding moiety. In order for the input strand to bind insufficiently to displace the output strand (i.e., for output strand displacement to be necessary for the input strand to extend along the gate strand), the output strand annealing recombination section and the input strand non-annealing recombination section at least partially overlap. In some forms, the output strand annealing recombination section and the input strand annealing recombination section at least partially overlap. However, too much overlap will induce displacement of the output strand merely by binding of the input strand to the exposed portion of the gate strand, without the need for polymerase-mediated extension of the input strand. Thus, any amount of overlap between the export strand annealing rebinding moiety and the import strand annealing rebinding moiety is less than an amount sufficient for the import strand to displace the export strand without polymerase-mediated extension. In a preferred form of the invention, the output strand annealing recombination section and the input strand annealing recombination section do not overlap. This is believed to allow for faster binding kinetics between the import and portal chains.
In some forms, the input strand annealing rebinding portion of the gate strand can serve as a primer for an amplification reaction, where the input strand annealing rebinding portion binds to a sequence on the template nucleic acid. In some forms, the amplification reaction is LAMP. The gate strand can serve as a Forward Inner Primer (FIP) or a reverse inner primer (BIP) in the LAMP reaction. Amplification of the forward outer primer (FP) and/or the backward outer primer (BP), respectively, can displace the output strand that was originally bound to the gate strand.
The polymerase dependent logic gate may be configured to perform any of the logical operations described herein, including being, not, and, or, nand, not, nor, exclusive or, exclusive nor, and implication operations, and the like.
An exemplary polymerase dependent or logic gate is shown in fig. 5. The input strand annealing recombination portion of gate strand 53 (green) has a first input strand annealing recombination portion (01 ×) and a second input strand annealing recombination portion (02 ×). The first import strand annealing rebinding moiety is substantially complementary to and anneals to the first import strand 51 (red). The second input strand annealing rebinding moiety (02) is substantially complementary to and anneals to the second input strand 52 (cyan). Annealing recombination of one or both of
FIG. 6 illustrates an exemplary polymerase dependent AND logic gate. This logic gate has a gate chain 63 (green) without pre-annealing recombination output chains, but instead employs in-situ generated output chains 64 (red/blue). Like an or gate, the input strand annealing recombination site (02 × 01) of the gate chain 63 has a first input strand annealing recombination site (01 ×) and a second input strand annealing recombination site (02 × 01). The first input strand annealing rebinding moiety (01 x) is substantially complementary to first input strand 61 (red) and anneals to rebinding to the first input strand. The second input strand annealing rebinding moiety (02) is substantially complementary to and anneals to the second input strand 62 (cyan). The export strand 64 is generated in situ by the binding of the first input strand 61 to the first input strand annealing recombination moiety and polymerase mediated extension of the first input strand 61 along the gate strand. Then, as the second input strand 62 is combined with the second input strand annealing recombination moiety and the second input strand is extended, the resulting output strand 64 is displaced to produce an extended input strand 65 (cyan/red/blue).
FIGS. 7A-7D illustrate alternative exemplary polymerase dependent and logic gates. This logic gate comprises at least two
Fig. 8 illustrates an exemplary polymerase dependent nand logic gate. This logic gate comprises at least two different chains 83, 84 (green, merry). The gate chains 83, 84 are each coupled to a different output chain 85, 86 (blue, red, respectively). Export strands 85, 86 are substantially complementary to each other and, once displaced from gate strands 83, 84, are capable of annealing and re-binding to each other. The input strand annealing recombination fraction of a first 83 (green) of the at least two different gate chains 83, 84 anneals to a first 81 (red/input 1) of the at least two different input chains 81, 82, but does not anneal to a second 82 (cyan/input 2) of the at least two different input chains 81, 82. The input chain annealing recombination section of the second 84 (merry red/input 2) of the at least two different gate chains 83, 84 anneals to the second 82 (merry red/input 2) of the at least two different input chains 81, 82, but does not anneal to the first 81 (cyan/input 2) of the at least two different input chains 81, 82. When only one 81 (cyan/in 2 or magenta/in 2) of the two different input chains 81, 82 is present, the output chain 85 or 86 (blue or red, respectively) is permuted and can be detected or used for signal passing in downstream logic gates. When both input chains 81, 82 (cyan/in 2 and magenta/in 2) are present, the permuted output chains 85, 86 anneal to re-bind to each other when released from the gate chains 83, 84 and isolate each other from detection or signal transfer in downstream logic gates.
FIGS. 9A-9D illustrate alternative exemplary polymerase dependent NAND logic gates. As shown in fig. 9A, this logic gate includes a chain of gates 116 (green) that includes sites (input 1) that are capable of binding to the first input chain 114 (red/input 1). The
FIG. 10 illustrates another exemplary polymerase dependent non-logic gate. This logic gate includes an activated output complex that includes reporter strand 118 (black) and gate strand 115 (blue) that serve as output strands. The gate strand 115 (blue) is substantially complementary to the input strand 119 (merry red) at the input portion. In its initial state, the
Exemplary lengths of the gate strands in the polymerase-dependent logic gate include lengths of about 4, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 or more nucleotide bases to about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300 or more nucleotide bases.
Exemplary lengths of the output strand annealing recombination sections of the gate strands in the polymerase-dependent logic gates include lengths of about 2, about 4, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 or more nucleotide bases to about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300 or more nucleotide bases.
Exemplary lengths of the output strand non-annealing recombination segments of the gate strands in the polymerase-dependent logic gates include lengths of about 1, about 2, about 4, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 or more nucleotide bases to about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300 or more nucleotide bases.
Exemplary lengths of input strand annealing recombination moieties for gate strands in a polymerase-dependent logic gate include lengths of about 1, about 2, about 4, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 or more nucleotide bases to about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300 or more nucleotide bases.
Exemplary lengths of the output strands in the polymerase dependent logic gate include lengths of about 2, about 4, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 or more nucleotide bases to about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300 or more nucleotide bases.
The input strand in any polymerase dependent logic gate may comprise an amplified nucleic acid or may comprise the output strand from an upstream logic gate in the loop. For example, when LAMP is used, the input strand may include a portion of the LAMP amplification product, such as a LAMP dumbbell product. For example, the 3' portion of the LAMP dumbbell product can bind to the import strand annealing heavy binding portion of the gate strand. A schematic representation of this mechanism is shown in fig. 11, where the LAMP dumbbell 91 serves as the input strand. The 3' portion of the LAMP dumbbell 91 binds to and extends along the gate strand 92 to produce an extended input strand 93, displacing the output strand 94 from the gate strand 92.
The output strand in any polymerase dependent logic gate may comprise the input strand of a downstream logic gate or the input strand of a reporter gate.
Reporter gates configured to detect the output strand may include branch migration mediated strand displacement reporter or polymerase dependent reporter gates as shown in fig. 2. By placing a fluorophore on the gate strand 42 (e.g., at the 5 'end) and a quencher on the output strand 43 (e.g., at the 3' end) of the polymerase dependent logic gate as shown in FIG. 4, a polymerase dependent reporter gate can be generated, thereby generating a reporter strand and a quencher strand. Alternatively, a polymerase dependent reporter gate may be generated by placing a fluorophore on the output strand 43 (e.g., at the 5 'end) of a polymerase dependent logic gate and a quencher on the gate strand 42 (e.g., at the 3' end) as shown in fig. 4, thereby generating a reporter strand and a quencher strand. In either case, binding of the input strand and polymerase-mediated extension displaces the quencher strand from the reporter strand and allows fluorescence to be detected from the fluorophore.
As used herein, "identical" refers to identical nucleic acid sequences. "substantially identical" refers to nucleic acid sequences that are identical or sufficiently identical to bind to the same third nucleic acid sequence under conditions suitable for enzyme-dependent isothermal amplification, such as loop-mediated isothermal amplification (LAMP). Thus, substantially the same encompasses the same, and any embodiment described herein as substantially the same may therefore be the same. "complementary" refers to a nucleic acid sequence with perfect base pairing. "substantially complementary" refers to nucleic acid sequences that have perfect base pairing (complementarity) or sufficient base pairing to bind to each other under conditions suitable for enzyme-dependent isothermal amplification (e.g., LAMP). Thus, substantial complementarity encompasses complementarity, and any embodiment described herein as substantially complementary can therefore be complementary (perfect base pairing). In any of the embodiments described herein, identity, substantial identity, complementarity, or substantial identity may occur over a length of 2-200 to bases or more, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 14, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at, At least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, or at least 190 to at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 28, at least 30, or more preferably at least 20, at least 24, or more preferably at least 24, or more than 20, At least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, or more bases.
In the methods described herein, separation can occur prior to lysis, lysis can occur prior to amplification, and/or amplification can occur prior to detection. In a preferred form, the droplets maintain a substantially constant volume throughout and between lysis, amplification and detection. In a preferred form, lysis, amplification and detection all occur without diluting the droplets after separation, without adding additional reagents to the droplets and/or without removing reagents or liquids from the droplets.
In some forms, the lysis, amplification, and/or molecular logic calculations are performed at substantially the same temperature. "substantially the same temperature" refers to a temperature range spanning about 50 ℃, about 45 ℃, about 40 ℃, about 35 ℃, about 30 ℃, about 25 ℃, about 20 ℃, about 15 ℃, about 10 ℃, about 5 ℃ or less.
In some forms, the lysis, amplification and/or molecular logic calculations are performed at a substantially constant temperature. By "substantially constant temperature" is meant that the temperature is maintained over time within a range of about 50 ℃, about 45 ℃, about 40 ℃, about 35 ℃, about 30 ℃, about 25 ℃, about 20 ℃, about 15 ℃, about 10 ℃, about 5 ℃ or less.
In some forms, a substantially constant temperature is maintained throughout and between lysis, amplification, and/or performing molecular logic calculations.
In some forms, the lysis, amplification, and molecular logic calculations are performed at a temperature of about 45 ℃, about 50 ℃, about 55 ℃, or about 60 ℃ to about 70 ℃, about 75 ℃, about 80 ℃, about 85 ℃, or about 90 ℃.
The methods described herein can be used to profile various aspects of a nucleic acid composition of a single cell using RNA or DNA of the cell as a nucleic acid template. For example, these methods can be used for expression profiling of expression patterns of mRNA or miRNA characterizing cells. Certain cell types, such as Circulating Tumor Cells (CTCs) or cells with specific lineages, have distinguishable expression patterns. See, e.g., Sieuwerts et al, 2011. The methods of the invention can be used to differentiate CTCs from non-CTCs, or for cells of one lineage from cells of another lineage. The methods of the invention may also be used to detect certain genetic mutations in a cell, such as Single Nucleotide Polymorphisms (SNPs) or other types of mutations. For the design of suitable LAMP primers for the detection of SNPs, see, e.g., Yongkiettrakul et al, 2016; and badalo et al, 2012.
Fig. 12 illustrates an
In some forms, the
Fig. 13 illustrates an exemplary device 113 capable of sorting droplets. This device comprises a continuous network of microfluidic channels 129 suitable for handling droplets, and a set of liquid electrodes 123, 124 capable of conducting electricity. The droplets first flow into the injection port 120 and are separated by re-injection of oil through the further port 121. After measuring the fluorescence of the droplet by one or more lasers 130 and one or more detectors 131, the droplet passes through the sorting junction 122. Based on the fluorescence of the droplets, the droplets are guided to the sort outlet 126 or the waste outlet 127 by applying an electric field via the liquid electrodes 123 and 124. Intermittent application of such a field generates a dielectrophoretic force on the droplets, thereby deflecting the droplets to the sort outlet 126. Sorting is fine-tuned by the amplitude and frequency of the DC current applied between the reference electrode 123 and the sorting electrode 124. The separation is also fine tuned by the application of Bias Oil (Bias Oil) through inlet 125. The pressure reducing diverter 128 balances the oil pressure between the sort port 126 and the waste outlet 127, thereby preventing pressure fluctuations that could disrupt sorting.
The system of the present invention comprises any combination of elements described herein for performing the method steps described herein.
The elements and method steps described herein may be used in any combination, whether or not explicitly described.
All combinations of method steps as used herein can be performed in any order, unless otherwise indicated or clearly contradicted by context in which the combination is referenced.
As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.
As used herein, numerical ranges are intended to include each number and subset of numbers subsumed within the range whether or not specifically disclosed. Further, these numerical ranges should be construed as supporting claims directed to any number or subset of numbers within the range. For example, a disclosure from 1 to 10 should be interpreted to support a range from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
All patents, patent publications, and peer review publications (i.e., "references") cited herein are expressly incorporated herein by reference to the same extent as if each individual reference were specifically and individually indicated to be incorporated by reference. In the event of a conflict between the present disclosure and an incorporated reference, the present disclosure should control.
It is to be understood that the invention is not limited to the specific configurations and arrangements of parts herein shown and described, but includes such modified forms thereof as fall within the scope of the appended claims.
Examples of the invention
Background
The following examples illustrate aspects of the invention for profiling nucleic acid compositions of each individual cell in a population of hematopoietic cells to detect Circulating Tumor Cells (CTCs) in the hematopoietic cells.
During cancer progression, cells are isolated from the primary tumor and spread throughout the bloodstream. These CTCs serve as valuable biomarkers for real-time monitoring of carcinogenesis, and the methods of detecting them described herein provide key tools in cancer detection and treatment.
Detection of CTCs remains a technical challenge because of their very low abundance in normal hematopoietic cells and a highly heterogeneous phenotype. The present invention provides a high throughput platform for single cell analysis that provides the ability to detect CTCs with high sensitivity and specificity.
CTC analysis platforms combine the specificity of molecular computing with the enormous throughput of droplet-based microfluidics. Single cells are encapsulated in droplets containing DNA-based logic circuits that input cell transcripts, perform logic calculations, and output fluorescent signals based on the phenotypic state of the cells (fig. 1). Droplets are generated on a microfluidic device capable of handling millions of cells per hour and incubated and analyzed. This approach is highly scalable and can be applied to tens of RNA inputs, thus providing a detailed map of the phenotypic status of the cells.
Materials and methods
Device fabrication
Microfluidic devices are made from Polydimethylsiloxane (PDMS) by a soft lithography process. SU-83025 or 3010 photoresist (Microchem) was deposited onto a silicon wafer and spun to obtain the desired layer height. A photomask is used to create a pattern of polymerized photoresist during UV exposure. The patterned wafer was then placed into a petri dish to form a mold and added at a polymer to activator ratio of 11:1
DNA complexes and primer mixtures
All DNA oligonucleotides were ordered from integrated DNA technology (corarville, iowa) using standard desalting techniques, except that the reporter complex strand was purified by HPLC. All LAMP primers were diluted in DNase/RNase-free water (Invitrogen, Cat. No.: 10977023) and stored at-20 ℃. The logic gate and reporter strand were diluted in 10mM phosphate buffered saline (pH 7.4) treated with DEPC and then stored at-20 ℃. A100 Xstock of each DNA complex was prepared in Phosphate Buffered Saline (PBS) treated with Diethylpyrocarbonate (DEPC) and stored at-20 ℃. On the day of the experiment, each DNA complex was annealed and rebinding separately by heating to 97 ℃ for 5 minutes and cooling to 23 ℃ at a rate of-2 ℃/min. The DNA complexes were stored on ice until the start of the experiment. A20 LAMP primer mix was prepared in DNase/RNase free water and stored at-20 ℃. The sequences of the primers and logic gates used in the LAMP experiments are shown in table 1.
Table 1: sequences of primers and logic gates used in LAMP experiments
TABLE 2 sequences of oligonucleotides used in CHA experiments
In vitro transcription
The DNA templates for KRT19 and VIM transcripts were synthesized by integrated DNA technology, Inc. (Colalville, Iowa) as "gBlocks" (SEQ ID NO:72 and SEQ ID NO:73, respectively) and cloned into pET-22b (+) vector under the T7 promoter (available from Novagen, Cat. No.: 69744-3). In vitro transcription was performed using HiScribeTMT7 high-yield RNA Synthesis kit (New England Biolabs, Cat: E2040S), and the obtained RNA was purified using the GeneJET RNA purification kit (Thermo Scientific, Cat: K0731). In NanoDropTMRNA concentration was quantified on a spectrophotometer (Seimer Feishell science Co., Ltd.) and the stock solution was incubated at-80 ℃Stored in DEPC-treated PBS.
Cell culture and staining
MOLT-4 cells (American Type Culture Collection) were subcultured every two days at a ratio of 1:8 and grown in RPMI-1640 medium (Gibco, Cat. No.: 11875093) supplemented with 10% FBS (Gibco, Cat. No.: 10082147) and 1X antibiotic antifungal (Gibco, Cat. No.: 15240062). SK-BR-3, U-2OS and MCF7 cells (American type culture Collection) were subcultured every two days at a ratio of 1:4 and grown in DMEM supplemented with 10% FBS and 1X antibiotic antifungal, high glucose (Gibco, Cat. No: 11965-092). On the day of the experiment, each cell type was collected and washed twice with Dulbecco's Phosphate Buffered Saline (DPBS) (Gibco, Cat. No.: 14190144). Then
Batch RT-LAMP experiment
Batch RT-LAMP assays were performed in triplicate at 65 ℃ on a CFX Connect from Bole. The reaction was carried out In a total volume of 10. mu.L, 1.6. mu.M for FIP/BIP primer, 0.2. mu.M for F3/B3 primer, 0.4. mu.M for LoopF/B primer, 1XWarmStart LAMP master mix (New England Biolabs, Cat. No.: E1700S), 0.5U/. mu.L/μ LSUPERAse. InTMRNase inhibitor (Invitrogen, Cat: AM2696), and 0.5X phosphate buffered saline. Different concentrations of the DNA complex were added. Table 1 shows LAMP primer and logic gate sequences. In the reaction without any DNA complex, LAMP fluorescent dye (new england biosciences) was added as a universal LAMP indicator. In lysate experiments1% Triton X-100 (Sigma Aldrich, Cat. No: T8787) was included in the reaction mixture. Intact, unstained cells were added to each well before the start of each experiment. For all experiments, the "standard concentration" of LAMP primers was defined as 1.6. mu.M per FIP/BIP primer, 0.2. mu.M per F3/B3 primer, and 0.4. mu.M per LoopF/B primer for a given LAMP primer set.
Transducer orthogonality experiment
The experiments were performed in triplicate as described above in the "batch RT-LAMP experiment" and slightly modified. Each reaction contains two LAMP primer sets: one specific for KRT19 transcript and the other specific for VIM transcript. Only one transducer was added per reaction to identify VIM or KRT19 amplification products. KRT19 or VIM RNA transcribed in vitro was added to each reaction at a concentration of 10 nM. PBS was also added to the non-template control reaction of each transducer. Each reaction contained a 400nM transducer gate strand pre-annealed to the 200nM transducer output strand, and a 200nM reporter quench strand pre-annealed to the 100nM reporter fluorophore strand. The logic gate sequences and LAMP primer sequences are shown in table 1.
AND logic gate experiments with LAMP input
The experiment was performed in triplicate similarly to the "batch RT-LAMP experiment". Each reaction contained both KRT19 primer set and VIM LAMP primer set at standard concentrations (as defined in the "batch RT-LAMP experiment"). In vitro transcribed KRT19 and/or VIM RNA was added at a concentration of 10 nM. Each reaction also contained 50nM RepF that previously annealed to re-bind to 100nM RepQ. Each reaction contains both KRT19 transducer and VIM transducer:
OR logic gate experiments with LAMP input
Each reaction contained standard concentrations (as defined in the batch RT-LAMP experiment) of the KRT19 primer set and vimlam primer set. In vitro transcribed KRT19 and/or VIM RNA was added at a concentration of 1nM, or an equivalent amount of water was used as a non-template control. Each reaction contained 100nM RefF preannealed to 200nM RepQ,
Logic gate experiments with LAMP input
Each reaction contained a standard concentration (as defined in the "batch RT-LAMP experiment") of the KRT19 primer set. In vitro transcribed KRT19 RNA was added at a concentration of 1nM, or an equivalent amount of water was used as a non-template control. Each reaction contained 100nM RePF pre-annealed to 200nM RepQ and
Non-logic gate experiments with LAMP input
Each reaction contained 200nM KRT19 and non-VIM export pre-annealed to 100nM RepF,
Nand logic gate experiments with LAMP input
Each reaction comprised a
Droplet RT-LAMP experiments with dsDNA-specific reporter
The experiments were performed as described above in the "droplet RT-LAMP experiments with fluorescent logic gate reporter" with some modifications. Prior to the experiments, MOLT-4 and SK-BR-3 cells were stained with calcein orange red AM (invitrogen), respectively, as described in "cell culture and staining" above. A co-flow microfluidic dropper with 100 μ M square channels was used to generate drops with a volume of about 1 nL. The drip maker consists of three inlets containing:
1.) Optiprep suspended at 18.75% v/vTM4X cell mixture (900 cells/. mu.L) in a mixture of density medium (Sigma Aldrich, Cat.: D1556) and phosphate buffered saline,
2.)2X WarmStart LAMP master mix (new england biosciences, catalog No.: E1700S) of the first and second,
3.) including LAMP primer, SUPERAse InTMRNase inhibitor (Invitrogen, Cat. No.: AM2696), 4XWarmStart LAMP fluorescent dye (New England Biolabs) and 4% v/v Triton X-100 (Sigma Aldrich, Cat. No.: T8787) in a 4X mixture.
These aqueous streams were combined into QX200 suspended in fluorinated oil (for EvaGreen, catalog # 1864005)TMDroplets create oil). The flow rates at
The final RT-LAMP conditions included 1.6. mu.M for FIP/BIP primer, 0.2. mu.M for F3/B3 primer, 0.4. mu.M for LoopF/B primer, 1XWarmStart LAMP master mix (New England Biolabs.), 0.5U/. mu.LSUPERAse. InTMRNase inhibitor (Invitrogen), 1% Triton X-100 (Sigma Aldrich), 4.7% v/v OptiprepTMDensity gradient media (sigma aldrich), 1X LAMP fluorescent dye (new england biosciences) and 0.5X phosphate buffer. Liquid drop RT-LAMP experiments with multiplex transducers
Cells were encapsulated with LAMP components, logic gates and lysis reagents using co-flow microfluidic drippers. Prior to the experiment, U-2OS and SK-BR-3 cells were stained with calcein AM (BD biosciences, Cat: 564061), respectively, as described above under "cell culture and staining". Selecting 60 μm as the width and height of the microfluidic channel, a droplet volume of approximately 250pL was produced. This dripper consists of three aqueous inlets containing:
1.) 10X cell mixture (5,000 cells/. mu.L) suspended in phosphate buffered saline,
2.)2X WarmStart LAMP master mix (new england biosciences, catalog No.: E1700S) of the first and second,
3.) including LAMP primer, DNA complex, SUPERAse InTMRNase inhibitor (Invitrogen, Cat.: AM2696) and 6.25% v/v Tween-20 (Sigma Aldrich, Cat.: P9416) in 2.5X mixtures.
These aqueous streams were combined into QX200 suspended in fluorinated oil (for EvaGreen, catalog # 1864005)TMDroplets create oil). The flow rate at
Each condition contained the KRT19 primer set and VIM primer set as listed in table 1. The final RT-LAMP conditions included 1.6. mu.M for each FIP/BIP primer, 0.2. mu.M for each F3/B3 primer, 0.4. mu.M for each LoopF/B primer, 100nM RepF-HEX strand pre-annealed to 200nM RepQ strand, 100nM RepF2-AF647 strand pre-annealed to 200nM RepQ2 strand, 100nM KRT19->110nM KRT 19-derived Rep transducer>Rep transducer gate chain, preannealing recombination to 100nM VIM->110nM VIM->Rep transductant, 1XWarmStart LAMP Master mix (New England Biolabs Inc.), 0.5U/. mu.L SUPERAse. InTMRNase inhibitor (Invitrogen) and 2.5% v/v Tween-20 (Sigma Aldrich, Cat. No.: P9416).
Droplet ESR1 RT-LAMP
The experiment was performed as "droplet RT-LAMP experiments with multiple transducers", with some modifications. MCF7 and SK-BR-3 cells were stained with calcein red-orange AM (invitrogen), respectively, as described in "cell culture and staining" above. Cells were loaded into the apparatus at a concentration of 9,000 cells/. mu.L in phosphate buffered saline containing 125 pg/. mu.L RNase A (Seimer Feishell technologies, Cat.: EN 0531). Each condition contained a standard concentration (as defined in the "batch RT-LAMP experiment") of ESR1 LAMP primer set. The ESR1 LAMP primer sequence is listed in table 1. A Warmstart LAMP dye (new england laboratories) was added to
Liquid droplet RT-LAMP with Integrated device
The experiment was performed as "droplet ESR1 RT-LAMP", with some modifications. MOLT-4 cells were stained with calcein red-orange AM (invitrogen) as described in "cell culture and staining" above. Cells were loaded into the device at a concentration of 9,000 cells/. mu.L in phosphate buffered saline. Each condition contained a standard concentration (as defined in the "batch RT-LAMP experiment") of GAPDH LAMP primer sets. The primer sequences are listed in table 1. The final RT-LAMP conditions consisted of a 1XWarmStart LAMP master mix (New England Biolabs), containing 1XWarmStart LAMP dye, 0.5U/. mu.LSUPERAse. InTMRNase inhibitor (Invitrogen) and 2.5% v/v Tween-20 (Sigma Aldrich, Cat. No.: P9416). The droplets were generated and incubated and analyzed in a single integrated device as shown in fig. 12. After extracting excess oil for tight droplet packing, the droplets flow out of the droplet generator into a section of PE tubing (Scientific models, inc., Lake havasu city, AZ)). The length of the PE tube was such that the droplets stayed in an incubator at 65 ℃ for 45 minutes and then were re-injected into the microfluidic device for fluorescence measurement. The droplets were excited with 473nm and 532nm lasers, and data were acquired and analyzed similarly to the above experiment. The flow rate of droplets produced at
Experiment of assembling catalytic hairpin
Catalytic Hairpin Assembly (CHA) was performed in a 10. mu.L reaction at 37 ℃ on a Tecan Spark plate reader. Fluorescence was measured with an excitation wavelength of 490nm and an emission wavelength of 535 nm. The reaction buffer consisted of TENAK (20mM Tris, 1mM EDTA, 140mM NaCl and 5mM KCl, pH 8.0) and 0.5% SDS. Each reaction contained a DNA oligonucleotide containing 50nM H1, 400nM H2, 50nM H3, 400nM H4, 10nM CK19 sensor and 50nM RepF-CHA that pre-annealed to re-bind to 100nM RepQ-CHA. The oligonucleotides were annealed and recombined as described in "DNA complexes and primer mixtures", respectively, except for RepF-CHA and RepQ-CHA, which were annealed and recombined together. The sequence of each oligonucleotide is given in table 2. For the CHA background experiment, reactions were run in triplicate with or without 100pM of DNA oligonucleotide "CK 19 input" to prime the reaction. The reaction was continued for 12 hours.
For lysate inhibition experiments, MOLT-4 cells were titrated into the LAMP reaction and final concentrations ranged from 3.9 x 105Cells/. mu.L to 1.0 x 108Cells/. mu.L. In the "CK 19 +" reaction, 10nM of DNA oligonucleotide "CK 19 input" was added to initiate the reaction, and if the "CK 19 input" was not added, no initiator was added. The reaction was continued for 8 hours.
Droplet sorting experiments
The droplet generation method was similar to that used in "droplet ESR1 RT-LAMP" above, with some modifications. MCF7 cells were stained with calcein red-orange AM (invitrogen) as described in "cell culture and staining" above, and SK-BR-3 cells were each stained with calcein violet AM (invitrogen). Cells were mixed at a ratio of 1:1 and loaded into the device at a total concentration of 9,000 cells/. mu.L in phosphate buffered saline. Each condition contained a standard concentration (as defined in the "batch RT-LAMP experiment") of ESR1 LAMP primer set. The primer sequences are listed in table 1. A Warmstart LAMP dye (new england laboratories) was added to
ACTB SNP-LAMP detection assay
The experiment was performed in a similar manner to the method used in the "batch RT-LAMP experiment" described above, with some modifications. Total RNA was extracted from MOLT-4 or SK-BR-3 cells using the GeneJET RNA purification kit (Seimer Feishell science, Cat: K0731) and added at a final concentration of 1 ng/. mu.L. ACTB LAMP primers were added at standard concentrations (as defined in the batch RT-LAMP experiment). In addition, 1. mu.M of the "ACTB 4B 3-library" strand was added. All oligonucleotide sequences are shown in table 1. The Warmstart LAMP dye (new england biosciences) was added as a universal LAMP indicator.
Results
DNA-based CTC detection circuit
The following example shows a DNA-based loop that inputs multiple cellular transcripts, performs logical calculations, and outputs a binary CTC classification. Early experiments showed that the DNA strand displacement cascade was not sufficient to detect transcript levels in single cells, and therefore required an input amplification step. Therefore, the development of amplification mechanisms sensitive, selective and resistant to high lysate concentrations is sought.
Signal amplification: a Catalytic Hairpin Assembly (CHA) amplification mechanism was initially explored. CHA uses input nucleic acids as catalysts to assemble metastable hairpins, resulting in linear signal amplification. CHA expansion was affected by a high signal background and strong inhibition of cell lysate (fig. 14A and 14B). After thorough characterization, it was concluded that CHA was not sufficient to amplify transcripts of single cells in microfluidic droplets. Hybridization Chain Reaction (HCR) was also tested as an amplification mechanism and similar results to CHA were found. In FIG. 14A, error bars represent +/-1 standard deviation of the mean. These experiments were performed as described in the "catalytic hairpin assembly experiments".
Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was identified as a robust technique for amplifying transcripts of single cells. LAMP uses six primers and a strand displacing DNA polymerase to exponentially amplify nucleic acid targets. By RT-LAMP, more than 10 were achieved6Sub-nanomolar detection sensitivity and lysate resistance at the target use concentration of individual cells/ml. RT-LAMP was tested for its ability to differentiate human breast cancer cells (SK-BR-3) from leukocytes (MOLT-4) using primers specific for the epithelial marker cytokeratin 19(CK19, KRT 19). These experiments revealed that the signal to background ratio>150 wide (50 min) detection window (fig. 15A and 15B). This excellent signal amplification is sufficient to feed downstream molecular logic gates and detect on microfluidic chips. Furthermore, it was determined that CK19 LAMP was even at 107SK-BR-3 and MOLT-4 lysates could also be reliably distinguished at each cell/ml (equivalent to one cell per 100pL droplet) (FIG. 15B). These experiments show that RT-LAMP on the epithelial marker KRT19 can distinguish human breast cancer cells (SK-BR-3) from leukocytes (MOLT-4) under conditions comparable to microdroplet reactions. Error bars represent +/-1 standard deviation of the mean for all experiments. These experiments were performed as described in the "batch RT-LAMP experiments".
Molecular logic: the CTC detection loop must integrate signals from multiple transcripts for CTC classification. DNA logic gates can perform complex molecular logic and calculations on nucleic acid inputs. A new logic gate was designed that utilizes the strand displacement activity of LAMP polymerase to drive strand displacement. This new mechanism provides better kinetics and less signal leakage than the traditional random walk branch migration cascade.
All basic single-input and two-input logic gates were designed using the polymerase-driven mechanism (fig. 4-11). In theory, an arbitrarily complex logic function can be computed by cascading these basic gates. As initial demonstrations, constructed were a gate (fig. 4) to detect the epithelial marker (KRT19), a not gate (fig. 10) to detect the mesenchymal marker (VIM), an or gate (fig. 7A-D) to identify the epithelial (KRT19) or mesenchymal (VIM) markers, an and gate (fig. 7A-7D) to identify the epithelial (KRT19) and mesenchymal (VIM) markers, and a nand gate (fig. 9A-D) to identify the epithelial (KRT19) and mesenchymal (VIM) markers. These gates are essential for identifying CTCs that occupy a transition state along epithelial-mesenchymal or mesenchymal-epithelial. Experiments on these gates demonstrated that strong and uniform fluorescence signals in the presence of KRT19 and/or VIM are consistent with the designed logic operation of the logic gate (fig. 16). Fig. 16 contains end-point results for the time traces shown for the not gate (fig. 19A), or gate (fig. 18A), and gate (fig. 18B), and nand gate (fig. 19B). These experiments were performed as described in "and logic gate experiment with LAMP input", "or logic gate experiment with LAMP input", "non-logic gate experiment with LAMP input", and "non-logic gate experiment with LAMP input". Error bars represent +/-1 standard deviation of the mean in all curves.
It has also been demonstrated that gates can be operated orthogonally to detect specific amplification events in multiplex LAMP responses. Experiments were performed using orthogonal, multiplex LAMP sum and logic gates that recognize VIM (fig. 17B) or KRT19 (fig. 17A), as described in the "transducer orthogonality experiments" above. Error bars represent +/-1 standard deviation of the mean.
Ultra-high flux microfluid CTC screening device
The following example provides an integrated microfluidic device that monitors the output of a DNA-based logic circuit developed in the experiment described above. These experiments demonstrate the ability to miniaturize the RT-LAMP reaction in microfluidic droplets and apply it to differentiate the phenotypic state of single cells.
Single cell analysis in microfluidic droplets: after optimization of LAMP conditions in a batch assay, it was reduced to a microemulsion format. Single cells were loaded onto a microfluidic device and encapsulated in approximately 1nL droplets containing lysis reagent, RT-LAMP reagent, and dsDNA-specific LAMP indicator. Cell staining (calcein red-orange AM, invitrogen) was used to verify co-localization of cells and LAMP signals. The drops were incubated for 20 minutes and imaged using fluorescent micrographs. White blood cells (MOLT-4) showed no KRT19 signal above background (FIG. 20B), while breast cancer cells (SK-BR-3) showed strong KRT19 expansion (FIG. 20A). This experiment demonstrates the ability to use LAMP in microemulsion droplets to differentiate cell types. This experiment was performed as described in "droplet RT-LAMP experiments with dsDNA specific reporter".
Further demonstrated is higher throughput single cell analysis using a full microfluidic workflow with continuous detection. The successful application of this technique to the analysis of estrogen receptor (ER, ESR1) expression in thousands of MCF7(ER +) and SK-BR-3(ER-) cells is shown in FIG. 21. As expected, a significantly larger fraction of MCF7 cells showed high fluorescence for LAMP indicators compared to SK-BR-3 cells. This indicates that the method employed can successfully distinguish cells in a high throughput manner based on their mRNA gene expression. This experiment was performed as described in "droplet ESR1 RT-LAMP".
It was also shown that multiple transcripts could be assayed simultaneously in these droplet assays, as shown in FIGS. 23A and 23B. A multiplex transducer experiment was performed in which KRT19 amplified an activated Alexa Fluor 647 reporter and VIM amplified an activated HEX reporter. SK-BR-3(CK19+/VIM-) and U-2OS (CK19-/VIM +) cells were encapsulated in droplets with these transducers and reporters, and each cell type was found to activate only its intended reporter. This experiment was performed as described in "droplet RT-LAMP experiments with multiple transducers".
Integrated microfluidic devices: all three microfluidic steps are combined into one continuous workflow without user intervention. This device comprises three modules. A module: (1) encapsulating the single cell in a droplet containing the DNA loop and amplification reagents; (2) incubating the reaction for a specified time and temperature; and (3) detecting the loop output using fluorescence spectroscopy. An exemplary system is shown in fig. 12. As shown in fig. 22, GAPDH transcripts can be amplified in thousands of MOLT-4 cells using this device. Based on these experiments, fully automated microfluidic devices can be fabricated that analyze millions of cells per hour. This experiment was performed as described in "droplet RT-LAMP with Integrated device".
Microfluidic droplet sorting: two cell types were separated based on expression of Estrogen Receptor (ER) transcript (ESR1) using a dielectrophoretic sorting device. MCF7(ER +) and SK-BR-3(ER-) cells were stained separately, mixed together and then sorted based on RT-LAMP amplification. As shown in fig. 24A and 24B, this device successfully enriched the green fluorescent droplet in the sort outlet, indicating that ESR1 amplification was more successful. This experiment demonstrates the ability to physically separate a cell population based on its transcriptional state. These sorted (pool) and unsorted pools can be further analyzed by RNA sequencing or other assays. Such sorting means are depicted in fig. 13. See also Scimambi and Abate, 2015.
Additional logic loops: the experiments outlined above demonstrate the ability to perform spectroscopic analysis of single cells in microfluidic droplets using strand displacement cascade. One advantage of molecular logic circuits is the ability to exchange new elements to adapt to the type of cancer and even the patient to make the determination. A logic loop can be constructed to profile multiple transcription inputs, such as a molecular marker information panel (information molecular marker panel) for a particular cancer type. The microfluidic device can be used to detect and/or classify low abundance CTCs in a human blood sample.
Additional sample and spectral analysis features: the assays and devices described herein may be used with samples other than blood samples. For example, cells from a tissue biopsy may be dispersed and subjected to the nucleic acid profiling described herein. Profiling can be used to profile any of a variety of nucleic acid features and classify cells in a variety of formats. Profiling the expression patterns of mRNA and/or miRNA and detecting or profiling SNPs provide only a few non-limiting examples. LAMP-based SNP detection has been shown to be feasible in droplets by differentiating MOLT-4 and SK-BR-3 total RNA. MOLT-4RNA containing SNPs in ACTB transcripts was amplified before SK-BR-3 ribonucleic acid. These results are shown in fig. 25. Error bars represent +/-1 standard deviation of the mean. This SNP assay was performed as described in the "ACTB SNP-LAMP detection assay".
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Yao D, Wang B, Xiao S, Song T, Huang F, Liang H, "what controls the" on/off "switch in the cohesive end-mediated strand displacement reaction on DNA-conjugated gold nanoparticles? (What Controls the "Off/on switch" in the tool hole-media string and display Reaction DNA joined gold Nanoparticles; 31(25):7055-61.
Yongkiettrakul S, Kampeera J, Chareanchim W, Rattacajak R, Porthanakemaem W, Kiatpatomochai W, Kongkasurichai D, "Simple detection of Plasmodium falciparum single nucleotide polymorphisms (Single detection of Single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMP assay in combination with lateral flow dipstick)", "International parasitology (Parasitiol Int)", 2 months 2017; 66(1):964-971.
Zanoli LM, Spoto G, "isothermal amplification method for the detection of nucleic acids in microfluidic devices" (biosensors (basels)), published as "biosensor (biosensors (basel)", 12/2012 and 27/s; 3(1):18-43. Zhang C, Yang J, Xu J, "Circular DNA logic gate with strand displacement (Circular DNA logic gate strand displacement)", "langmuir", 2 months and 2 days 2010; 26(3):1416-9.
Zhang H, Jenkins G, Zou Y, Zhu Z, Yang CJ, "Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics (Massively parallel single-molecule and single-cell emulsion conversion polymerase chain reaction)", "analytical chemistry,
Zhu J, Zhang L, Dong S, Wang E, "Four way junction-driven DNA strand displacement and its use in constructing most logic loops" (Four-way junction-drive DNA strand displacement and ligation in building bulk large logic circuit), "american chemical society-nano (ACSNano.), (26/11/2013); 7(11):10211-7.
Zou C, Wei X, Zhang Z, Liu C, Zhou C, Liu Y, "Four-fold simulation calculation Based on DNA Strand Displacement", proceedings of the American society of chemistry (ACS Omega), 20172 (8), 4143-.
Sequence listing
<110> Wisconsin alumi Research Foundation
<120> high throughput nucleic acid profiling of single cells
<130>09824097-P180094WO01
<150>US 62/639,822
<151>2018-03-07
<160>73
<170> PatentIn version 3.5
<210>1
<211>1921
<212>DNA
<213> Intelligent people
<400>1
gagtgagcgg cgcggggcca atcagcgtgc gccgttccga aagttgcctt ttatggctcg 60
agcggccgcg gcggcgccct ataaaaccca gcggcgcgac gcgccaccac cgccgagacc 120
gcgtccgccc cgcgagcaca gagcctcgcc tttgccgatc cgccgcccgt ccacacccgc 180
cgccagctca ccatggatga tgatatcgcc gcgctcgtcg tcgacaacgg ctccggcatg 240
tgcaaggccg gcttcgcggg cgacgatgcc ccccgggccg tcttcccctc catcgtgggg 300
cgccccaggc accagggcgt gatggtgggc atgggtcaga aggattccta tgtgggcgac 360
gaggcccaga gcaagagagg catcctcacc ctgaagtacc ccatcgagca cggcatcgtc 420
accaactggg acgacatgga gaaaatctgg caccacacct tctacaatga gctgcgtgtg 480
gctcccgagg agcaccccgt gctgctgacc gaggcccccc tgaaccccaa ggccaaccgc 540
gagaagatga cccagatcat gtttgagacc ttcaacaccc cagccatgta cgttgctatc 600
caggctgtgc tatccctgta cgcctctggc cgtaccactg gcatcgtgat ggactccggt 660
gacggggtca cccacactgt gcccatctac gaggggtatg ccctccccca tgccatcctg 720
cgtctggacc tggctggccg ggacctgact gactacctca tgaagatcct caccgagcgc 780
ggctacagct tcaccaccac ggccgagcgg gaaatcgtgc gtgacattaa ggagaagctg 840
tgctacgtcg ccctggactt cgagcaagag atggccacgg ctgcttccag ctcctccctg 900
gagaagagct acgagctgcc tgacggccag gtcatcacca ttggcaatga gcggttccgc 960
tgccctgagg cactcttcca gccttccttc ctgggcatgg agtcctgtgg catccacgaa 1020
actaccttca actccatcat gaagtgtgac gtggacatcc gcaaagacct gtacgccaac 1080
acagtgctgt ctggcggcac caccatgtac cctggcattg ccgacaggat gcagaaggag 1140
atcactgccc tggcacccag cacaatgaag atcaagatca ttgctcctcc tgagcgcaag 1200
tactccgtgt ggatcggcgg ctccatcctg gcctcgctgt ccaccttcca gcagatgtgg 1260
atcagcaagc aggagtatga cgagtccggc ccctccatcg tccaccgcaa atgcttctag 1320
gcggactatg acttagttgc gttacaccct ttcttgacaa aacctaactt gcgcagaaaa 1380
caagatgaga ttggcatggc tttatttgtt ttttttgttt tgttttggtt tttttttttt 1440
ttttggcttg actcaggatt taaaaactgg aacggtgaag gtgacagcag tcggttggag 1500
cgagcatccc ccaaagttca caatgtggcc gaggactttg attgcacatt gttgtttttt 1560
taatagtcat tccaaatatg agatgcgttg ttacaggaag tcccttgcca tcctaaaagc 1620
caccccactt ctctctaagg agaatggccc agtcctctcc caagtccaca caggggaggt 1680
gatagcattg ctttcgtgta aattatgtaa tgcaaaattt ttttaatctt cgccttaata 1740
cttttttatt ttgttttatt ttgaatgatg agccttcgtg cccccccttc cccctttttt 1800
gtcccccaac ttgagatgta tgaaggcttt tggtctccct gggagtgggt ggaggcagcc 1860
agggcttacc tgtacactga cttgagacca gttgaataaa agtgcacacc ttaaaaatga 1920
g 1921
<210>2
<211>1921
<212>DNA
<213> Intelligent people
<400>2
gagtgagcgg cgcggggcca atcagcgtgc gccgttccga aagttgcctt ttatggctcg 60
agcggccgcg gcggcgccct ataaaaccca gcggcgcgac gcgccaccac cgccgagacc 120
gcgtccgccc cgcgagcaca gagcctcgcc tttgccgatc cgccgcccgt ccacacccgc 180
cgccagctca ccatggatga tgatatcgcc gcgctcgtcg tcgacaacgg ctccggcatg 240
tgcaaggccg gcttcgcggg cgacgatgcc ccccgggccg tcttcccctc catcgtgggg 300
cgccccaggc accagggcgt gatggtgggc atgggtcaga aggattccta tgtgggcgac 360
gaggcccaga gcaagagagg catcctcacc ctgaagtacc ccatcgagca cggcatcgtc 420
accaactggg acgacatgga gaaaatctgg caccacacct tctacaatga gctgcgtgtg 480
gctcccgagg agcaccccgt gctgctgacc gaggcccccc tgaaccccaa ggccaaccgc 540
gagaagatga cccagatcat gtttgagacc ttcaacaccc cagccatgta cgttgctatc 600
caggctgtgc tatccctgta cgcctctggc cgtaccactg gcatcgtgat ggactccggt 660
gacggggtca cccacactgt gcccatctac gaggggtatg ccctccccca tgccatcctg 720
cgtctggacc tggctggccg ggacctgact gactacctca tgaagatcct caccgagcgc 780
ggctacagct tcaccaccac ggccgagcgg gaaatcgtgc gtgacattaa ggagaagctg 840
tgctacgtcg ccctggactt cgagcaagag atggccacgg ctgcttccag ctcctccctg 900
gagaagagct acgagctgcc tgacggccag gtcatcacca ttggcaatga gcggttccgc 960
tgccctgcgg cactcttcca gccttccttc ctgggcatgg agtcctgtgg catccacgaa 1020
actaccttca actccatcat gaagtgtgac gtggacatcc gcaaagacct gtacgccaac 1080
acagtgctgt ctggcggcac caccatgtac cctggcattg ccgacaggat gcagaaggag 1140
atcactgccc tggcacccag cacaatgaag atcaagatca ttgctcctcc tgagcgcaag 1200
tactccgtgt ggatcggcgg ctccatcctg gcctcgctgt ccaccttcca gcagatgtgg 1260
atcagcaagc aggagtatga cgagtccggc ccctccatcg tccaccgcaa atgcttctag 1320
gcggactatg acttagttgc gttacaccct ttcttgacaa aacctaactt gcgcagaaaa 1380
caagatgaga ttggcatggc tttatttgtt ttttttgttt tgttttggtt tttttttttt 1440
ttttggcttg actcaggatt taaaaactgg aacggtgaag gtgacagcag tcggttggag 1500
cgagcatccc ccaaagttca caatgtggcc gaggactttg attgcacatt gttgtttttt 1560
taatagtcat tccaaatatg agatgcgttg ttacaggaag tcccttgcca tcctaaaagc 1620
caccccactt ctctctaagg agaatggccc agtcctctcc caagtccaca caggggaggt 1680
gatagcattg ctttcgtgta aattatgtaa tgcaaaattt ttttaatctt cgccttaata 1740
cttttttatt ttgttttatt ttgaatgatg agccttcgtg cccccccttc cccctttttt 1800
gtcccccaac ttgagatgta tgaaggcttt tggtctccct gggagtgggt ggaggcagcc 1860
agggcttacc tgtacactga cttgagacca gttgaataaa agtgcacacc ttaaaaatga 1920
g 1921
<210>3
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> primer KRT19 LAMP-
<400>3
agtgacatgc gaagccaat 19
<210>4
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> primer KRT19 LAMP-
<400>4
gctttcatgc tcagctgtga 20
<210>5
<211>41
<212>DNA
<213> Artificial sequence
<220>
<223> primer KRT19 LAMP-3 FIP
<400>5
agcgacctcc cggttcaatt ctcgagcaga accggaagga t 41
<210>6
<211>41
<212>DNA
<213> Artificial sequence
<220>
<223>KRT19 LAMP-3 BIP
<400>6
cacacggagc agctccagat gtgcagctca atctcaagac c 41
<210>7
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> primer KRT19 LAMP-3 LF
<400>7
tggtgaacca ggcttcagc 19
<210>8
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> primer KRT19 LAMP-3 LB
<400>8
aggtccgagg ttactgacct gc 22
<210>9
<211>17
<212>DNA
<213> Artificial sequence
<220>
<223> primer VIM LAMP-
<400>9
ccgcaccaac gagaagg 17
<210>10
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> primer VIM LAMP-
<400>10
tggttagctg gtccacct 18
<210>11
<211>38
<212>DNA
<213> Artificial sequence
<220>
<223> primer VIM LAMP-2 FIP
<400>11
tccaggaagc gcaccttgtc ggagctgcag gagctgaa 38
<210>12
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> primer VIM LAMP-2 BIP
<400>12
aagatcctgc tggccgagct cccgcatctc ctcctcgtag 40
<210>13
<211>17
<212>DNA
<213> Artificial sequence
<220>
<223> primer VIM LAMP-2 LF
<400>13
agttggcgaa gcggtca 17
<210>14
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> primer VIM LAMP-2 LB
<400>14
cagctcaagg gccaaggcaa 20
<210>15
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> primer ESR1 LAMP-
<400>15
agagctgcca acctttgg 18
<210>16
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> primer ESR1 LAMP-
<400>16
tgaaccagct ccctgtctg 19
<210>17
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> primer ESR1 LAMP-1 FIP
<400>17
ggcactgacc atctggtcgg aagcccgctc atgatcaaac 40
<210>18
<211>40
<212>DNA
<213> Artificial sequence
<220>
<223> primer ESR1 LAMP-1 BIP
<400>18
ttgttggatg ctgagccccc cccatcatcg aagcttcact 40
<210>19
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> primer ESR1 LAMP-1 LF
<400>19
gccaggctgt tcttcttaga gc 22
<210>20
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> primer ESR1 LAMP-1 LB
<400>20
actctattcc gagtatgatc ctacc 25
<210>21
<211>16
<212>DNA
<213> Artificial sequence
<220>
<223> primer GAPDH LAMP-
<400>21
gctgccaagg ctgtgg 16
<210>22
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> primer GAPDH LAMP-
<400>22
cccaggatgc ccttgagg 18
<210>23
<211>41
<212>DNA
<213> Artificial sequence
<220>
<223> primer GAPDH LAMP-2 FIP
<400>23
gttggcagtg gggacacgga acaaggtcat ccctgagctg a 41
<210>24
<211>38
<212>DNA
<213> Artificial sequence
<220>
<223> primer GAPDH LAMP-2 BIP
<400>24
tgtcagtggt ggacctgacc tgtccgacgc ctgcttca 38
<210>25
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> primer GAPDH LAMP-2 LF
<400>25
ggccatgcca gtgagctt 18
<210>26
<211>25
<212>DNA
<213> Artificial sequence
<220>
<223> primer GAPDH LAMP-2 LB
<400>26
cgtctagaaa aacctgccaa atatg 25
<210>27
<211>17
<212>DNA
<213> Artificial sequence
<220>
<223> primer ACTB 4B 3-SNP
<400>27
ggctggaaga gtgccgc 17
<210>28
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>28
gcggctacag cttcacca 18
<210>29
<211>42
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>29
cgtggccatc tcttgctcga aggggaaatc gtgcgtgaca tt 42
<210>30
<211>38
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>30
gcttccagct cctccctgga ccgctcattg ccaatggt 38
<210>31
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>31
acgtagcaca gcttctcctt 20
<210>32
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>32
gaagagctac gagctgcct 19
<210>33
<211>17
<212>DNA
<213> Artificial sequence
<220>
<223> primer ACTB 4B 3-pool
<400>33
gcggcactct tccagcc 17
<210>34
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> reporter RePF
<400>34
cgagtgctgc gtatgacaag ggctagcgtt 30
<210>35
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> reporter RePF-HEX
<400>35
cgagtgctgc gtatgacaag ggctagcgtt 30
<210>36
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> reporter RepQ
<400>36
cccttgtcat acgcagcact cg 22
<210>37
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> reporter RePF2-AF647
<400>37
cgccgcgtcc tgatctaact gactgactgc 30
<210>38
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223> reporter RepQ2
<400>38
tcagttagat caggacgcgg cg 22
<210>39
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19- > Rep transductant gate
<400>39
cgagtgctgc gtatgacaag ggctagcgtt atgctacgag cgacctcccg gttcaattct 60
<210>40
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19- > Rep transducer output
<400>40
aacgctagcc cttgtcatac gcagcactcg 30
<210>41
<211>58
<212>DNA
<213> Artificial sequence
<220>
<223> VIM- > Rep transductant gate
<400>41
cgagtgctgc gtatgacaag ggctagcgtt atgctacgtc caggaagcgc accttgtc 58
<210>42
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> VIM- > Rep transducer output
<400>42
aacgctagcc cttgtcatac gcagcactcg 30
<210>43
<211>88
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and
<400>43
cgagtgctgc gtatgacaag ggctagcgtt atgctacgtc caggaagcgc accttgtcat 60
gctacgagcg acctcccggt tcaattct 88
<210>44
<211>88
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and
<400>44
cgagtgctgc gtatgacaag ggctagcgtt atgctacgag cgacctcccg gttcaattct 60
atgctacgtc caggaagcgc accttgtc 88
<210>45
<211>88
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 or VIM gate
<400>45
cgagtgctgc gtatgacaag ggctagcgtt atgctacgtc caggaagcgc accttgtcat 60
gctacgagcg acctcccggt tcaattct 88
<210>46
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 or VIM output
<400>46
aacgctagcc cttgtcatac gcagcactcg 30
<210>47
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223>VIM F1
<400>47
gacaaggtgc gcttcctgga 20
<210>48
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223>KRT19 F1
<400>48
agaattgaac cgggaggtcg ct 22
<210>49
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>49
ctgctctcac ggaggcgcac cggtaagggt catcgatgag cgacctcccg gttcaattct 60
<210>50
<211>58
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>50
ctgctctcac ggaggcgcac cggtaagggt catcgatgtc caggaagcgc accttgtc 58
<210>51
<211>35
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19/
<400>51
cgatgaccct taccggtgcg cctccgtgag agcag 35
<210>52
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and VIM gate
<400>52
cgagtgctgc gtatgacaag ggctagcgtt atgctgctct cacgg 45
<210>53
<211>30
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and VIM outputs
<400>53
aacgctagcc cttgtcatac gcagcactcg 30
<210>54
<211>50
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and VIM threshold
<400>54
ccgctggtga tcactctgct ctcacggagg cgcaccggta agggtcatcg 50
<210>55
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>55
cgcgatccga gtgctgcgta tgacaagggc tagcgtttgc cggaagcgac ctcccggttc 60
<210>56
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>56
cgcgatccga gtgctgcgta tgacaagggc tagcgtttgc cggatccagg aagcgcacct 60
<210>57
<211>44
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19/
<400>57
tccggcaaac gctagccctt gtcatacgca gcactcggat cgcg 44
<210>58
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>58
ccatcgcgga gacacggaca tcgttaaggc agcctgtagg cagcctccag gaagcgcacc 60
<210>59
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223>
<400>59
gtgtctccgc gatggcgagt gctgcgtatg acaagggcta gcgttagcga cctcccggtt 60
<210>60
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and non-VIM inhibitors
<400>60
ggctgcctac aggctgcctt aacgatgtcc gtgtctccgc gatgg 45
<210>61
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19 and non-VIM outputs
<400>61
aacgctagcc cttgtcatac gcagcactcg ccatcgcgga gacac 45
<210>62
<211>60
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19- > Rep2 transducin gate
<400>62
gtgtctccgc gatggcgccg cgtcctgatc taactgactg actgcagcga cctcccggtt 60
<210>63
<211>45
<212>DNA
<213> Artificial sequence
<220>
<223> KRT19- > Rep2 transducer output
<400>63
gcagtcagtc agttagatca ggacgcggcg ccatcgcgga gacac 45
<210>64
<211>55
<212>DNA
<213> Artificial sequence
<220>
<223> CK19 inductor
<400>64
gagttaccag cctggagttc tcaatggtgg cctggtaact cactgaccga gctaa 55
<210>65
<211>67
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide H1
<400>65
cgacatctaa cctagctcac tgaccgagct aagctgttct cgattagctc ggtcagtgag 60
ttaccag 67
<210>66
<211>46
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide H2
<400>66
gctgttctcg atcactgacc gagctaatcg agaacagctt agctcg 46
<210>67
<211>67
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide H3
<400>67
gtcagtgagc taggttagat gtcgccatgt gtagacgaca tctaacctag cccttgtcat 60
agagcac 67
<210>68
<211>46
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide H4
<400>68
agatgtcgtc tacacatggc gacatctaac ctagcccatg tgtaga 46
<210>69
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide RepF-CHA
<400>69
cgagtgctct atgacaaggg ctaggtt 27
<210>70
<211>20
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide RepQ-CHA
<400>70
cccttgtcat agagcactcg 20
<210>71
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> oligonucleotide CK19 input
<400>71
gccaccattg agaactccag g 21
<210>72
<211>1496
<212>DNA
<213> Artificial sequence
<220>
<223>KRT19 gBlock
<400>72
caggtctcgt atgagatatc cgcccctgac accattcctc ccttcccccc tccaccggcc 60
gcgggcataa aaggcgccag gtgagggcct cgccgctcct cccgcgaatc gcagcttctg 120
agaccagggt tgctccgtcc gtgctccgcc tcgccatgac ttcctacagc tatcgccagt 180
cgtcggccac gtcgtccttc ggaggcctgg gcggcggctc cgtgcgtttt gggccggggg 240
tcgcctttcg cgcgcccagc attcacgggg gctccggcgg ccgcggcgta tccgtgtcct 300
ccgcccgctt tgtgtcctcg tcctcctcgg gggcctacgg cggcggctac ggcggcgtcc 360
tgaccgcgtc cgacgggctg ctggcgggca acgagaagct aaccatgcag aacctcaacg 420
accgcctggc ctcctacctg gacaaggtgc gcgccctgga ggcggccaac ggcgagctag 480
aggtgaagat ccgcgactgg taccagaagc aggggcctgg gccctcccgc gactacagcc 540
actactacac gaccatccag gacctgcggg acaagattct tggtgccacc attgagaact 600
ccaggattgt cctgcagatc gacaatgccc gtctggctgc agatgacttc cgaaccaagt 660
ttgagacgga acaggctctg cgcatgagcg tggaggccga catcaacggc ctgcgcaggg 720
tgctggatga gctgaccctg gccaggaccg acctggagat gcagatcgaa ggcctgaagg 780
aagagctggc ctacctgaag aagaaccatg aggaggaaat cagtacgctg aggggccaag 840
tgggaggcca ggtcagtgtg gaggtggatt ccgctccggg caccgatctc gccaagatcc 900
tgagtgacat gcgaagccaa tatgaggtca tggccgagca gaaccggaag gatgctgaag 960
cctggttcac cagccggact gaagaattga accgggaggt cgctggccac acggagcagc 1020
tccagatgag caggtccgag gttactgacc tgcggcgcac ccttcagggt cttgagattg 1080
agctgcagtc acagctgagc atgaaagctg ccttggaaga cacactggca gaaacggagg 1140
cgcgctttgg agcccagctg gcgcatatcc aggcgctgat cagcggtatt gaagcccagc 1200
tgggcgatgt gcgagctgat agtgagcggc agaatcagga gtaccagcgg ctcatggaca 1260
tcaagtcgcg gctggagcag gagattgcca cctaccgcag cctgctcgag ggacaggaag 1320
atcactacaa caatttgtct gcctccaagg tcctctgagg cagcaggctc tggggcttct 1380
gctgtccttt ggagggtgtc ttctgggtag agggatggga aggaagggac ccttaccccc 1440
ggctcttctc ctgacctgcc aataaaaatt tatggtccaa gggtgagcga gaccac 1496
<210>73
<211>706
<212>DNA
<213> Artificial sequence
<220>
<223>VIM gBlock
<400>73
caggtctcgt atgccaccca cacccaccgc gccctcgttc gcctcttctc cgggagccag 60
tccgcgccac cgccgccgcc caggccatcg ccaccctccg cagccatgtc caccaggtcc 120
gtgtcctcgt cctcctaccg caggatgttc ggcggcccgg gcaccgcgag ccggccgagc 180
tccagccgga gctacgtgac tacgtccacc cgcacctaca gcctgggcag cgcgctgcgc 240
cccagcacca gccgcagcct ctacgcctcg tccccgggcg gcgtgtatgc cacgcgctcc 300
tctgccgtgc gcctgcggag cagcgtgccc ggggtgcggc tcctgcagga ctcggtggac 360
ttctcgctgg ccgacgccat caacaccgag ttcaagaaca cccgcaccaa cgagaaggtg 420
gagctgcagg agctgaatga ccgcttcgcc aactacatcg acaaggtgcg cttcctggag 480
cagcagaata agatcctgct ggccgagctc gagcagctca agggccaagg caagtcgcgc 540
ctgggggacc tctacgagga ggagatgcgg gagctgcgcc ggcaggtgga ccagctaacc 600
aacgacaaag cccgcgtcga ggtggagcgc gacaacctgg ccgaggacat catgcgcctc 660
cgggagaaat tgcaggagga gatgcttcag agatgagcga gaccac 706
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