Genotyping of mutations by in-tube hybridization in combination with universal tag-microarray
阅读说明：本技术 通过管内杂交与通用标签-微阵列的组合的突变的基因分型 (Genotyping of mutations by in-tube hybridization in combination with universal tag-microarray ) 是由 玛赛拉·基亚里 弗朗西斯科·达明 西尔维亚·加尔比亚蒂 莫里齐奥·费拉里 于 2018-03-30 设计创作，主要内容包括：目标基因片段的测定方法,其包括：扩增至少一种包含或可能包含至少一种目标SNP区域的目标基因片段,以形成初始扩增产物；从初始扩增产物形成至少一种单链扩增产物,其中所述单链扩增产物包含或可能包含至少一种目标SNP区域；在溶液中将所述单链扩增产物与至少一种报道分子杂交,所述报道分子包含寡核苷酸的至少两个不同的结构域,其中寡核苷酸的第一结构域用于与单链扩增产物杂交,并且其中寡核苷酸的第二结构域用于与至少一种微阵列探针表面杂交,其中微阵列探针表面包含至少一种捕获探针以允许杂交；将杂交单链扩增产物的溶液与至少一种微阵列探针表面接触,所述微阵列探针表面包含至少一种捕获探针以允许杂交；检测微阵列表面上杂交单链扩增产物的存在。在与癌症基因分型相关的优选实施方案中,KRAS癌基因的出人意料的良好结果表明所研究的所有7种密码子12和13突变可在小于90分钟内在组织临床样品中明确检测到。此外,该系统可以揭示代表少于0.1％起始材料的突变等位基因。通过将突变检测与阵列杂交解偶联,该技术变得通用。(A method for assaying a target gene fragment, comprising: amplifying at least one target gene fragment comprising or potentially comprising at least one SNP region of interest to form an initial amplification product; forming at least one single-stranded amplification product from the initial amplification product, wherein the single-stranded amplification product comprises or may comprise at least one SNP region of interest; hybridizing the single-stranded amplification product in solution with at least one reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of an oligonucleotide is used for hybridization with the single-stranded amplification product, and wherein a second domain of an oligonucleotide is used for hybridization with at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow hybridization; contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization; detecting the presence of the hybridized single stranded amplification product on the surface of the microarray. In a preferred embodiment associated with cancer genotyping, unexpectedly good results for the KRAS oncogene indicate that all 7 codon 12 and 13 mutations studied can be unambiguously detected in clinical samples of tissue in less than 90 minutes. In addition, the system can reveal mutant alleles that represent less than 0.1% of the starting material. This technique becomes versatile by uncoupling the mutation detection from the array hybridization.)
1. A method for assaying a target gene fragment, comprising:
amplifying at least one target gene fragment comprising or potentially comprising at least one SNP region of interest to form an initial amplification product;
forming at least one single-stranded amplification product from the initial amplification product, wherein the single-stranded amplification product comprises or may comprise at least one SNP region of interest;
hybridizing the single-stranded amplification product in solution with at least one reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of an oligonucleotide is used for hybridization with the single-stranded amplification product, and wherein a second domain of an oligonucleotide is used for hybridization with at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the microarray surface.
2. A method for assaying a target gene fragment, comprising:
amplifying at least one target gene fragment comprising or potentially comprising at least one SNP region of interest to form an initial amplification product, wherein the fragment to be amplified is primed in the amplification to enable (1) separation of one single strand of the initial amplification product from another single strand in a subsequent step of the assay, and (2) detection of the separated single strands in a subsequent step of the assay;
separating at least one single-stranded amplification product from the initial amplification product, wherein the single-stranded amplification product comprises or may comprise at least one SNP region of interest;
hybridizing the single-stranded amplification product to at least one reporter molecule comprising at least two different domains of an oligonucleotide in solution, wherein a first domain of the oligonucleotide is for hybridizing to the single-stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the microarray surface.
3. A method of determining a target KRAS, NRAS, BRAF and/or PIK3CA oncogene fragment comprising:
amplifying at least one target KRAS, NRAS, BRAF and/or PIK3CA oncogene fragment comprising or potentially comprising at least one target SNP region to form an initial amplification product;
forming at least one single-stranded amplification product from the initial amplification product using denaturation and coupling to beads, wherein the single-stranded amplification product comprises or may comprise at least one SNP region of interest, wherein optionally the isolated single-stranded amplification product is stabilized prior to further hybridization;
hybridizing the single-stranded amplification product in solution with at least one reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of an oligonucleotide is used for hybridization with the single-stranded amplification product, and wherein a second domain of an oligonucleotide is used for hybridization with at least one microarray probe surface, and wherein the hybridizing step comprises applying a temperature gradient covering at least two different hybridization temperatures, and wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the microarray surface with fluorescence.
4. A method of determining a target KRAS, NRAS, BRAF and/or PIK3CA oncogene fragment comprising:
amplifying at least one target KRAS, NRAS, BRAF and/or PIK3CA oncogene fragment comprising or potentially comprising at least one target SNP region to form an initial amplification product, wherein the fragment to be amplified is primed in the amplification to enable (1) separation of one single strand of the initial amplification product from another single strand by coupling to beads in a subsequent step of the assay, and (2) detection of the separated single strands by fluorescence detection in a subsequent step of the assay;
separating at least one single stranded amplification product from the initial amplification product using denaturation and coupling to beads, wherein the single stranded amplification product comprises or may comprise at least one SNP region of interest, wherein optionally the separated single stranded amplification product is stabilized prior to further hybridization;
hybridizing the single-stranded amplification product with at least one reporter molecule in solution, said reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of an oligonucleotide is used for hybridization with the single-stranded amplification product, and wherein a second domain of an oligonucleotide is used for hybridization with at least one microarray probe surface, and wherein the hybridizing step comprises applying a temperature gradient covering at least two different hybridization temperatures, and wherein said microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the microarray surface with fluorescence.
5. The assay of claim 1 or 3, wherein the fragments to be amplified are primed in amplification to enable detection of the isolated single strands in a subsequent step of the assay.
6. The assay of claim 1 or 3, wherein the fragments to be amplified are primed in amplification to enable (1) separation of one single strand of the initial amplification product from the other single strand in a subsequent step of the assay, and (2) detection of the separated single strands in a subsequent step of the assay.
7. The assay of claim 1 or 3, wherein forming at least one single-stranded amplification product from the initial amplification product comprises isolating a single-stranded amplification product, wherein the single-stranded amplification product comprises or is likely to comprise at least one SNP region of interest.
8. The assay of any of claims 1-2, wherein the assay and gene fragment of interest are used to detect at least one single mutation in the KRAS, NRAS, BRAF and/or PIK3CA oncogenes.
9. The assay of any one of claims 1-8, wherein the assay and target gene fragment are used to detect at least one single mutation in the KRAS oncogene.
10. The assay of any one of claims 1-9, wherein the assay and gene fragment of interest are used to detect at least one KRAS oncogene mutation at codon 12, and/or codon 13, and/or codon 61, and/or codon 146.
11. The assay of any one of claims 1-10, wherein the assay and gene fragment of interest are used to detect at least one KRAS oncogene mutation at codon 12 and/or codon 13.
12. The assay of any one of claims 1-11, wherein the assay and target gene fragment are for detecting at least one KRAS oncogene mutation comprising at least one of the following mutations: G12A, G12C, G12D, G12R, G12S and/or G12V in codon 12; and/or G13D in codon 13; and/or Q61HC Q61HT, Q61L, Q61R and/or Q61K in codon 61; and/or a146T in codon 146.
13. The assay of any one of claims 1-8, wherein the assay and target gene fragment are used to detect at least one single mutation in the NRAS oncogene.
14. The assay of any one of claims 1-8 or 13, wherein the assay and gene fragment of interest are used to detect at least one NRAS oncogene mutation at codon 12 and/or codon 13.
15. The assay of any one of claims 1-8 or 13-14, wherein the assay and target gene fragments are used to detect at least one NRAS oncogene mutation comprising at least one of the following mutations: G12A, G12C, G12D, G12S, G12V in codon 12 and/or G13D, G13R, G13V in codon 13.
16. The assay of any of claims 1-8, wherein the assay and target gene fragment are used to detect at least one single mutation in the BRAF oncogene.
17. The assay of any of claims 1-8 or 16, wherein the assay and gene segment of interest are used to detect at least one BRAF mutation at codon 600.
18. The assay of any of claims 1-8 or 16-17, wherein the assay and gene segment of interest are used to detect a BRAF oncogene mutation, which is a V600E mutation in codon 600.
19. The assay of any one of claims 1-8, wherein the assay and the gene segment of interest are used to detect at least one single mutation in the PIK3CA oncogene.
20. The assay of any of claims 1-8 or 19, wherein the assay and gene fragment of interest are used to detect at least one PIK3CA mutation at codons 542, 545, and/or 1047.
21. The assay of any of claims 1-8 or 19-20, wherein the assay and gene fragment of interest are used to detect at least one PIK3CA mutation comprising at least one of an E542K mutation in codon 542, an E545K mutation in codon 545, and/or an H1047R mutation in codon 1047.
22. The assay of any one of claims 1-21, wherein the initial amplification products are linked to magnetic beads to enable separation of one single strand of the initial amplification products from another single strand in a subsequent step of the assay.
23. The assay of any one of claims 1-22, wherein the initial amplification product attached to the beads is isolated by centrifugation or filtration.
24. The assay of any one of claims 1-23, wherein the initial amplification product is linked to a tag that enables fluorescent detection of the isolated single strands in a subsequent step of the assay.
25. The assay of any one of claims 1-24, wherein the initial amplification product is attached to particles that enable interferometric detection of the isolated single strands in subsequent steps of the assay.
26. The assay of any one of claims 1-25, wherein the amplifying step comprises PCR amplification.
27. The assay of any one of claims 1-26, wherein the separating step comprises thermal denaturation of the initial amplification product and separation of one single strand from the other single strand.
28. The assay of any one of claims 1-27, wherein the separating step comprises chemical denaturation of the initial amplification product and separation of one single strand from the other single strand.
29. The assay of any one of claims 1-28, wherein the hybridizing step comprises applying a temperature gradient covering at least two different hybridization temperatures.
30. The assay of any one of claims 1-29 wherein the hybridizing step comprises hybridization of a wild-type sequence.
31. The assay of any one of claims 1-30, wherein the isolated single stranded amplification product is stabilized prior to further hybridization.
32. The assay of any of claims 1-31, wherein the conformation of the isolated single stranded amplification product is stabilized by hybridization to an oligonucleotide whose sequence is adjacent to a sequence comprising a mutation prior to further hybridization.
33. The assay of any of claims 1-32 wherein the microarray probe surface comprises at least seven different capture probes.
34. The assay of any of claims 1-33, wherein the microarray probe surface comprises at least one terpolymer coating on the surface of the substrate to which the capture probes are bound.
35. The assay of any of claims 1-34, wherein the microarray probe surface comprises at least one terpolymer coating on the substrate surface bound to the capture probes, and the terpolymer coating comprises: at least one polymer backbone unit that is acrylamide or methacrylamide; at least one second polymeric backbone unit adapted to bind to the substrate surface; and at least one third polymeric backbone unit bound to the capture probe.
36. The assay of any of claims 1-35, wherein the microarray probe surface comprises at least one terpolymer coating on the substrate surface bound to the capture probes, and wherein the copolymer is further blocked to prevent non-specific binding during the assay.
37. The assay of any one of claims 1-36 wherein the detecting step comprises at least one colorimetric, chemiluminescent, label-free detection by SPR or interference, or fluorescent detection step.
38. The assay of any one of claims 1-37, wherein the detecting step is a fluorescence detecting step.
39. A kit adapted to perform any of the assay method steps of any of claims 1-38.
40. A kit adapted to perform all assay method steps of any one of claims 1-38.
Brief Description of Drawings
Fig. 1-15 and tables 1, 2, 3, and 4 further illustrate preferred embodiments described and/or claimed in greater detail below. Any colors used in the filed application, including the filed drawings, form part of the original disclosure and may be relied upon for enforceability, written description and statement.
FIG. 1. assay protocol. (A) The single-stranded PCR product containing the SNP region is hybridized with two oligonucleotides called "reporters" whose sequence consists of two parts (domains), one complementary to the potentially mutated sequence of the gene and the other complementary to the oligonucleotides immobilized on the surface of the microarray slide, called "barcodes". Mutant and wild-type reporters are associated with two different barcode sequences. In the hybridization conditions used, only reporters (wild-type or mutated) that are perfectly complementary to the PCR product form hybrids. In the case of figure 1, the sample contains only KRAS wild type alleles, so only specific wild type reporters hybridize in solution to single stranded PCR products (ssPCR), whereas specific mutant reporters (variation positions circled and red) do not.
After hybridization has occurred, the solution contacts the microarray surface, where oligonucleotides complementary to the barcode sequence are spotted at specific locations. As shown in fig. 1 (B), the different barcodes in the 3' portion of the reporter sequence (barcode W for the wild-type allele, barcode M for the mutant allele) capture their complementary sequences. However, only the barcode sequence bound to the reporter that has formed a hybrid with PCR became fluorescent, as only the PCR fragment was tagged at the 5' end with a sequence called U-TAG, which interacts with the complementary Cy 3-labeled oligonucleotide Universal-Cy 3 (U-Cy 3) added in the final step of the assay.
FIG. 2 is a representative sequence of operating steps in an assay having the time required for the steps. The total time was 87 minutes.
FIG. 3.8 fluorescence images of microarray slides, each spotted with 8 barcode sequences (one for wild type and seven for mutant sequences) for genotyping seven (G12A, G12C, G12D, G12R, G12S, G12V and G13D), more frequently KRAS mutations. This experiment was intended to demonstrate that each mutant sequence was captured at a specific position of the array and that there was no cross-talk with other barcode sequences.
(A) Schematic representation of spotted barcode probe array. A silicon chip coated with copolymehzed (DMA-NAS-MAPS) was used as a substrate for covalently attaching amino-modified barcode probe oligonucleotides spotted at discrete locations. Each position in the 2X 4 grid identifies a single barcode probe address (and corresponding KRAS mutant or wild-type sequence).
(B) Each slide was hybridized with either wild-type or mutated single-stranded PCR fragments, previously incubated in solution with all eight different reporters. The barcode associated with a particular reporter drives the construct to the correct position on the array surface. Fluorescence detection was obtained by incubating the array with a universal Cy 3-labeled oligonucleotide complementary to the tagged reverse primer of single-stranded PCR. G12A, C, D, R, S, V and G13D correspond to control samples containing the indicated mutations. All 8 samples of known genotypes (wild type or mutant) were correctly identified.
FIG. 4 detection limits of G12D KRAS mutations. The sensitivity of the system was assessed by serial dilution (5%; 2.5%; 1.25%; 0.62%; 0.31%; 0.075%; 0.037%) of mutant DNA with appropriate mixing of wild-type DNA.
(A) Schematic representation of spotted barcode probe arrays for sensitivity assays. Wt = barcode probe oligonucleotide address of wild type single stranded PCR; G12D = barcode probe oligonucleotide address of G12D KRAS mutant single stranded PCR.
(B) The graph shows the relative fluorescence intensity corresponding to the signal of Cy 3-labeled mutant single-stranded PCR bound to a G12D barcode probe. The fluorescence intensity of the signal corresponding to Cy 3-labeled wild-type single stranded PCR bound to the wild-type barcode probe is not shown to simplify the histogram. All columns are the average of the intensity of 36 spots (6 x6 subarrays) of the G12D barcode probe array. WT = background fluorescence was present on the G12D barcode probe array of the chip hybridized to the wild type control sample. Error bars are the standard deviation of fluorescence intensity for each sample. Asterisks indicate the results of unpaired t-tests of different dilution curve points related to WT controls. ns = not significant. The significance is as follows: p < 0.05; p < 0.05; p < 0.01; -p = p < 0.001; p < 0.0001.
FIG. 5 genotyping of KRAS mutations in Formalin Fixed Paraffin Embedded (FFPE) samples. The spotting protocol for barcode sequences was the same as in fig. 3A. A graph of the relative fluorescence intensity of eight control clinical samples after hybridization to eight spotted chips is presented. G12A, C, D, R, S, V and G13D indicate the FFEP genotype. All columns are the average of the intensity of 36 spots (6 x6 subarrays) of each barcode probe subarray. Error bars are the standard deviation of fluorescence intensity for each sample.
FIG. 6 microarray images for analysis of G12S mutant samples. (A) Cy3 fluorescence image obtained after hybridization of single-stranded PCR-reporter complexes to universal tag array. By universal is meant that all mutant or wild type PCR fragments are detected with the same oligonucleotide. (B) Schematic representation of spotted barcode sequences. Each position in the 2X 4 grid identifies a single barcode probe address (and corresponding KRAS mutant or wild-type sequence). (C) Graph of relative fluorescence intensity. All columns are the average of the intensity of 36 spots (6 x6 subarrays) of each barcode probe subarray. Error bars are the standard deviation of fluorescence intensity for each sample.
In FIGS. 7-11, for sub-embodiments, two portions of the reporter are bound to a core structure such as a synthetic polymer or macromolecular scaffold. This increases the number of reporters and barcodes on the reporter and may increase sensitivity.
FIG. 7. examples of polymeric, or macromolecular scaffolds (cores) that bind to reporter and barcode sequences of a reporter molecule include dendrimer cores, and linear polymer cores. The core is surface functionalized.
FIG. 8 scheme for derivatization of azido dendrimers: the DBCO-modified oligonucleotide is added to a solution of the azido dendrimer. In particular, the oligonucleotides complementary to the PCR (wild type or mutant barcode) are 4 times as many oligonucleotides as complementary to the spotted probe (specific reporter).
FIG. 9. mutant PCR hybridization protocol with dendrimers modified with sequences complementary to the mutant sequences of the PCR.
FIG. 10 azido dendrimers modified with mutant oligonucleotides hybridize to mutant PCR and recognize mutant probes on the surface.
Fig. 11. (a) spotting protocol: wild type (left array) and mutant barcode probes (right array) were spotted on each chip at 10 μ M in 150mM sodium phosphate pH8.5, 0.01% sucrose monolaurate; (b) fluorescence images after hybridization with the dendrimer modified with Wt PCR (chip A) and the dendrimer modified with Mut PCR (chip B) and (c) fluorescence intensity signals.
FIG. 12. extended sequence set: (A) schematic representation of spotted barcode probe array. (B) The microarray scanned the Cy3 fluorescence signals from five different silicon chips.
Fig. 13 shows the lower detection limit: using a 16-well incubation chamber, mixtures of wild-type and mutant DNA in different ratios were incubated in parallel on different wells of the same slides (A) and (B). (C) An example of a calibration curve showing the KRAS G12S mutation. Table (D) reports the detection limit for each mutation.
FIG. 14. to evaluate the applicability of the tag-microarray approach in a real clinical setting, FFPE samples were analyzed. Typical results are shown in fig. 14 for samples identified with (a) KRAS G12S, (B) KRAS Q61HC mutation, and (C) NRAS G12V mutation.
Fig. 15 to evaluate the applicability of the tag-microarray approach in liquid biopsies, 3 ctDNA samples were analyzed. The array was used for liquid biopsy to analyze cell-free circulating tumor dna (ctdna) in plasma extracted from colorectal cancer patients, and the results of microarray analysis are shown in fig. 15, and their correctness was confirmed by droplet digital PCR.
The figure shows a typical fluorescence image of an array used to detect one of the 22 mutations reported in the protocol. ctDNA, 2 mutants and 1 wild type were analyzed in 3 patients. Although there is always a wild type signal present (upper left), only mutated DNA produces a signal in the yellow square. From the intensity of these signals, the concentration of the mutant DNA can be estimated by a calibration curve.
Attached table:
TABLE 1 sequence of KRAS mutated deposition probes and reporters.
TABLE 2 sequence of BRAF mutated deposition probes and reporters.
TABLE 3 sequence of PIK3CA mutant spotting probes and reporters.
TABLE 4 sequence of NRAS mutated deposition probes and reporters.
Detailed Description
Introduction to
Additional embodiments, including working examples, are provided in the detailed description below.
U.S. priority provisional application, U.S. serial No. 62/479995 filed on 31/3/2017, incorporated herein by reference in its entirety for all purposes.
This document provides a list of cited references later. No admission is made that any of these references or any other references cited in this document are actually prior art.
As supported by existing laws, this specification includes all conjunctions, whether open or closed, including "comprising," "consisting essentially of," and "consisting of," and including if used in the preamble of a claim or for a particular element of a claim. Thus, for example, if an embodiment is described and/or claimed using "comprising," that embodiment may also be written with "consisting essentially of" or "consisting of instead of" comprising. The phrase "consisting essentially of" limits the scope of the claims or elements to the specified materials or steps of the claimed invention or elements "as well as those having no material impact on the basic and novel features.
In a preferred embodiment, a PCR/LDR (polymerase chain reaction/ligase detection reaction) step is not used in the assay (see, e.g., Favis et al,Annals New York Academy of Sciences906,39-43 (2000) for PCR/LDR). In a preferred embodiment, the enzyme is not used in the assay. In a preferred embodiment, the reporter molecule is not immobilized on the surface.
The rationale for nucleic acid and genomic science is known in the art. See, for example, Calladine et al,Understanding DNA，The Molecule and How it Workssecond edition, 1997; the number of Gibson et al,A Primer of Genome Science,2002. For genotyping and microarrays in the patent literature, see, e.g., WO 2011/080068; WO 01/21838; WO 00/61801; WO/65098; WO 01/25485; WO 98/28438; and U.S. patent publication 2005/0244860. This includes knowledge about single and double stranded DNA, oligonucleotides, separating nucleic acid molecules and polymers from cells, and complementary sequences capable of hybridizing to each other.
A variety of genetic sources can be studied by the assay methods described herein, including human genetic sources (cell-free circulating DNA taken from human tissue or extracted from plasma). The assay may be part of disease diagnosis, disease prognosis, disease prevention and prevention procedures, and disease therapy and treatment. The person (and tissue taken from the person) undergoing the assay may or may not have been diagnosed with the disease. Preferred targets for the assays of the invention are human cancers and DNA applied to tissues removed from human cancer patients or DNA circulating in the blood. Of course, the assay can be more generally used for animals and their diseases, if desired.
The five steps in the assay, including the amplification step, the separation step, the hybridization step, the contacting step, and the detection step, are described in more detail below. Steps in larger assay methods such as hybridization, contact, and detection are known in the art (see, e.g., U.S. patent No. 6,806,047).
FIG. 2 illustrates an embodiment showing a series of steps after amplification of a target fragment. These include: (1) capture PCR magnetic beads, (2) PCR denaturation, (3) incubation of PCR and stabilizer, (4) incubation of PCR-stabilizer and reporter in temperature gradient, (5) hybridization of PCR-stabilizer-reporter on chip and detection with U-Cy3, and (6) final wash step.
In one aspect, there is provided a method for determining a target gene fragment, comprising:
amplifying at least one target gene fragment comprising at least one target SNP region to form an initial amplification product, wherein the fragment to be amplified is primed in the amplification to enable (1) separation of one single strand of the initial amplification product from another single strand in a subsequent step of the assay, and (2) detection of the separated single strands in the subsequent step of the assay;
separating at least one single-stranded amplification product from the initial amplification product, wherein the single-stranded amplification product contains or may contain at least one SNP region of interest;
hybridizing the single-stranded amplification product with at least one reporter molecule in solution, said reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of the oligonucleotide is for hybridizing to the single-stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the surface of the microarray.
A more specific aspect is an assay method for a target KRAS, NRAS, BRAF and/or PIK3CA oncogene fragment comprising:
amplifying at least one target KRAS, NRAS, BRAF and/or PIK3CA oncogene fragment comprising or potentially comprising at least one target SNP region to form an initial amplification product, wherein the fragment to be amplified is primed in the amplification to enable (1) separation of one single strand of the initial amplification product from another single strand by coupling to beads in a subsequent step of the assay, and (2) detection of the separated single strands by fluorescence detection in a subsequent step of the assay;
separating at least one single stranded amplification product from the initial amplification product using denaturation and coupling to beads, wherein the single stranded amplification product comprises or may comprise at least one SNP region of interest, wherein the separated single stranded amplification product is optionally stabilized prior to further hybridization;
hybridizing the single-stranded amplification product with at least one reporter molecule comprising at least two different domains of an oligonucleotide in solution, wherein a first domain of the oligonucleotide is for hybridizing to the single-stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to at least one microarray probe surface, and wherein the hybridizing step comprises applying a temperature gradient covering at least two different hybridization temperatures, and wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
the presence of the hybridized single stranded amplification product on the microarray surface is detected by fluorescence.
Additional embodiments are described in more detail below.
Step of amplification
Amplification methods for gene fragments are known in the art, in particular PCR amplification. Amplification may be performed with single-stranded or double-stranded nucleic acids. Manipulation and sequencing of amplification products is also known. See, for example, Bevan et al, "Sequencing of PCR-amplified DNA"PCR Methods and ApplicationsPage 222-228 (1992); T.A. Brown, a high-frequency,Gene Cloning and DNA Analysis: An Introduction: version 7, 2016, contains a description of PCR.
A wide range of target gene fragments can be used, and the KRAS, BRAF, PIK3CA and NRAS genes are preferred embodiments. As known in the art, an amplification step can produce an "initial amplification product. This initial amplification product may then be converted into a single stranded amplification product, which is subjected to further assay steps. The number of base pairs in the fragment to be amplified is not particularly limited, but may be, for example, 50bp to 300bp, or 100bp to 200 bp.
"target gene fragments" are known in the art and may include or possibly include one or more SNPs and/or SNP regions known in the art.
The gene fragment of interest may be "primed" as known in the art in forming the initial amplification product. Forward and reverse primers are known in the art. Priming may be performed in such a way that the fragment to be amplified is primed in the amplification to provide (1) separation of one single strand of the initial amplification product from the other single strand in a subsequent step of the assay, and (2) detection of the separated single strands in a subsequent step of the assay.
For example, priming may be performed using primers that are part of a specific binding pair (e.g., biotin and streptavidin) as known in the art for affinity capture.
Priming may also be performed so that the product can be labelled with moieties that allow detection.
In some embodiments, the assays and target gene fragments are used to detect at least one single mutation in the KRAS, NRAS, BRAF and/or PIK3CA oncogenes. In some embodiments, the assay and the target gene fragment are used to detect at least one single mutation in the KRAS oncogene. In some embodiments, the assays and target gene fragments are used to detect at least one KRAS oncogene mutation at
As known to those skilled in the art, one gene, oncogene, SNP and/or mutation may be tested.
In some embodiments, the initial amplification products are linked to, for example, particles or beads (e.g., magnetic beads) to enable separation of one single strand of the initial amplification products from another single strand in a subsequent step of the assay. In some embodiments, the initial amplification product is linked to a tag that enables fluorescent detection of the isolated single strands, e.g., in a subsequent step of the assay. In some embodiments, the amplifying step comprises PCR amplification.
Separation step
In some embodiments, a separation step may be performed. In such embodiments, the initial amplification product may then be subjected to a separation step in which the single stranded amplification product is separated, and the other single strand may be discarded or used for other purposes. For example, one strand can be bound to a solid phase such as a particle, including a magnetic particle, using affinity capture and specific binding. After binding, the bound strand can be separated from the target single strand for further use in assays.
In some embodiments, the separating step comprises heat denaturation of the initial amplification product and separation of one single strand from the other single strand. In some embodiments, the hybridizing step comprises applying a temperature gradient covering at least two different hybridization temperatures. In some embodiments, the hybridizing step comprises hybridization of a wild-type sequence. In some embodiments, the isolated single stranded amplification product is stabilized prior to further hybridization.
One embodiment provides amplification products that are combined with beads or particles and separated, for example, by centrifugation.
In other embodiments, no separation step is performed.
Hybridization step
The single stranded amplification product, which has been suitably primed and isolated (purified), is then subjected to a hybridization step in solution with at least one reporter molecule. The reporter comprises at least two different domains of an oligonucleotide, wherein a first domain of the oligonucleotide is for hybridizing to a single stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to a surface of at least one microarray probe. One reporter may comprise only one first domain and only one second domain. In addition, one reporter may comprise two or more first domains and/or two or more second domains. Sensitivity can be increased by using more domains per reporter.
A plurality of different reporters may be used. Thus, multiplexing can be performed, and the number of different reporters is not particularly limited. The number may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
In one embodiment, the reporter is a single oligonucleotide that contains two distinct domains, a first domain and a second domain. In another embodiment, a vector or core is used, wherein the first and second domains are different oligonucleotides linked to the vector or core. Here, the reporter molecule comprises a macromolecule or polymer conjugated or covalently bonded to the first and second domains. The macromolecule may be, for example, a water-soluble polymer, a dendrimer, or a dendrimer, as described further below. Water soluble polymers, dendrimers and dendrimers are known in the art. Water-soluble polymers, dendrimers and dendrimers can be highly branched to form cores, which can be surface functionalized with first and second domains.
Examples of reporter sequences (or domains) are shown in tables 1-4 for four mutations, including a first domain and a second domain, but other reporter sequences can be used to meet the need. This includes sequences that hybridize to the single-stranded amplification products, as well as sequences designed to hybridize to the spotted capture probes ("tails of the reporter").
In a preferred embodiment, the hybridization step comprises applying a temperature gradient covering at least two different hybridization temperatures. Temperature gradients for controlling hybridization are known in the art (see, e.g., U.S. patent publication 2005/0221367). The hybridization step may comprise applying a temperature gradient covering at least two, three, four, five, six or seven (for example) different hybridization temperatures, if more than one oligonucleotide reporter is used. A linear or stepwise gradient may be used. For example, a stepwise gradient may be used, holding the solution at a particular temperature for, e.g., 1-20 minutes, or 1-10 minutes, or about 5 minutes. Table 1 below shows examples of specific hybridization temperatures that can be used for particular sequences, but other hybridization temperatures and sequences can also be used. Hybridization temperature can be varied according to factors known in the art such as salt concentration. The hybridization temperature may be any known hybridization temperature. The hybridization temperature may be, for example, in the range of 25 ℃ to 65 ℃,27 ℃ to 55 ℃, 28 ℃ to 45 ℃, or 29 ℃ to 41 ℃.
In a preferred embodiment, the hybridizing step comprises hybridization of the wild type sequence.
In a preferred embodiment, the isolated single stranded amplification product is stabilized prior to hybridization. More specifically, the conformation of the isolated single stranded amplification product can be stabilized by hybridization with an oligonucleotide having a sequence complementary to a region adjacent to the region complementary to the reporter, which oligonucleotide can be referred to as a stabilizer. The stabilizing agent may be an oligonucleotide necessary to open secondary structures present in the amplicon prior to further hybridization with the reporter. Tables 1-4 show examples of stabilizer sequences, but other stabilizers may also be used.
Step of contacting
The solution comprising the hybridized reporter can then be contacted with at least one microarray probe surface comprising at least one capture probe. Microarrays are generally known in the art. See, for example, Kohane et al,Microarrays for an Integrative Genomics2003; the result of the process is that in Mueller et al,Microarrays, 2006。
the microarray may comprise at least three elements, and the elements may be processed into a microarray by methods known in the art.
As known in the art, the first element is a solid microarray substrate, which may for example be made of an inorganic material, and may for example be a silicon chip or a glass slide.
As is known in the art, the second component is a coating that may be applied to a substrate. The coating may be a thin film coating less than 100 microns thick. The coating may be based on one or more polymers, such as copolymers and terpolymers, having a plurality of repeating units with different functions. The majority of the repeating units may be, for example, acrylamide or methacrylamide monomers. A small number of repeat units may allow for further binding. In one function, the coating polymer and its coating may covalently bind one or more biomolecules, including capture probes. For example, the coating can have repeating units with electrophilic groups (e.g., NAS, N-acryloyloxy succinimide) that react with nucleophilic groups such as amino groups. The coating may also have repeating units that are reactive or better able to bind to the substrate surface, such as reactive silane groups. In addition, after the coating immobilizes the desired biomolecule (e.g., capture probe), the coating can be "blocked" with blocking moieties known in the art, such as amino compounds, including ethanolamine, to eliminate residual functional groups in the coating that would interfere with the assay and promote non-specific binding. Coatings for immobilizing biomolecules and coating of substrates for arrays are described, for example, in Chiari
The third element is one or more capture probes, typically different capture probes, which are biomolecules suitable for binding to the coating on the substrate and binding to and hybridizing to the portion of the solution in contact with the microarray surface. The capture probes may be used in the form of arrays or spots as known in the art. The capture probes may be applied to the coating by spotting methods known in the art to form the desired pattern and array. The capture probe may be an oligonucleotide that is functionalized to react with the coating polymer. The number of capture probes may be adapted to the particular assay.
In another embodiment, the capture probes or oligonucleotides are bound to particles and the hybridization events are detected by a flow cytometer of the type used in Luminex systems.
The spotted capture probes described in tables 1-4 are examples only, and other probes may be used to meet the needs of the assay.
In some embodiments, the microarray probe surface comprises at least seven different capture probes and probes for capturing wild-type moieties.
In some embodiments, the microarray probe surface comprises at least one terpolymer coating on the substrate surface bound to the capture probes. In some embodiments, the microarray probe surface comprises at least one terpolymer coating on the substrate surface bound to the capture probes, and the terpolymer coating comprises: at least one polymer backbone unit that is acrylamide or methacrylamide; at least one second polymeric backbone unit adapted to bind to a substrate surface; and at least one third polymeric backbone unit bound to the capture probe. In some embodiments, the microarray probe surface comprises at least one terpolymer coating on the substrate surface bound to the capture probes, and wherein the copolymer is further blocked to prevent non-specific binding during the assay.
Detection step
The detection step may also be performed to provide informative information about the gene segment of interest. Detection can be carried out by known methods including, for example, fluorimetry, colorimetry, chemiluminescence, or label-free methods by surface plasmon resonance or interferometry. As is known in the art, fluorescent dyes can be used for fluorescence detection.
In some embodiments, the detecting step comprises at least one colorimetric, chemiluminescent, label-free detection by SPR or interference, or fluorescent detection step. Methods known in the art may be used for detection, including, for example, the use of fluorophores, or particles such as gold particles, or enzymes for colorimetric detection. In some embodiments, the detecting step is a fluorescence detecting step.
Tables 1-4 show examples of sequences that can be used to bind the dye (known as universal-Cy 3), but other sequences and dyes can be used. In another embodiment, the universal tag carries biotin, which can be used to bind, for example, fluorescent streptavidin, streptavidin-modified gold particles, or other types of particles.
Reagent kit
One or more kits may be provided to carry out the assay methods of the invention, as is known in the art. For example, instructions for using the kit can be provided. One or more oligonucleotides may be provided as part of a kit. One or more reporter molecules may be provided in the kit. The kit may also be considered a system for performing an assay. In one embodiment, a kit is provided that is suitable for performing any or all of the assay method steps described and/or claimed herein. Various sub-combinations of steps may be performed with the kit.
Performance and advantages of the assay
A number of individual performance advantages, as well as combinations of performance advantages, are described and demonstrated herein with respect to at least some embodiments.
One particular advantage is the speed or rapidity of the measurement. See, for example, fig. 2 and working examples. The assay may be performed in less than, for example, 240 minutes, or less than 180 minutes, or less than 120 minutes, or less than 90 minutes after the amplification step. As technology advances, better instruments and methods are developed and automation of use is used, with no specific absolute lower limit on time. The lower limit may be, for example, 10 minutes, 20 minutes, or 30 minutes.
Another particular advantage is the sensitivity and detection limit of the assay. See, for example, working examples and figure 4. The detection limit can be reduced to below 0.1%, or reduced to below 0.75%, or reduced to below 037%. With the advancement of technology, better instruments and methods and automation of use were developed, with no specific absolute lower limit on the detection limit.
Working examples
Preferred embodiments are described below by way of non-limiting working examples.
1. Results
1.1. Overview of the method
The assay protocol for the preferred embodiment is shown in figure 1. The 167-bp fragment was amplified using 5 '-biotin forward and 5' -tagged reverse primers. Biotin allows binding of double stranded PCR products to streptavidin coated magnetic beads, while the tag sequence on the reverse primer (universal tag, U-tag) allows detection of single stranded PCR by hybridization to a fluorescent oligonucleotide (universal-Cy 3). After amplification, biotinylated double stranded PCR products were bound to streptavidin coated magnetic beads and captured on a magnet. The bead-modified PCR fragments are released in solution. After heat denaturation, the single-stranded DNA with the U-tag in the supernatant was recovered while the DNA strand bound to the magnetic beads was captured with a magnet and discarded. ssDNA hybridizes in solution with eight specific oligonucleotide reporters whose sequence consists of two domains. The 5 'domain corresponds to the KRAS sequence (wild-type or mutant reporter) while the 3' domain is a "barcode" sequence that identifies the oligonucleotide probe (barcode probe) spotted at a specific location on the silicon chip. ssDNA hybridized to a specific reporter is captured on the array surface by hybridization to the barcode region. Single stranded PCR products captured at specific positions corresponding to a given mutation were revealed by incubating the silicon chip with a universal Cy 3-oligonucleotide, the sequence of which is complementary to the U-tag present at the 5' end of the single stranded PCR.
1.2. Optimizing genotyping
Kirstein RAS (KRAS) is the most common mutant proto-oncogene, which is critical for tumor progression. KRAS mutations occur early in the tumorigenic pathway, and thus detection of KRAS mutations can be used for early diagnosis, prognosis, and assessment of treatment outcome in cancer treatment29. KRAS is an effector molecule of Epidermal Growth Factor Receptor (EGFR), a key target for the design of therapeutic strategies for the treatment of metastatic colorectal cancer (CRC). Fundamentally, activating mutations at
In addition to KRAS mutations, changes in DNA sequence (e.g., somatic point mutations) are the most common class of variants associated with the development of solid tumors, and span common oncogenic events, such as NRAS, PIK3CA, and BRAF mutations.
Classical methods used in microarray genotyping based on spotting specific capture sequences at different positions of the microarray fail to detect a few point mutations. High analytical sensitivity and specificity are required when a small number of mutant sequences are present in a large background of wild type. In classical SNP microarray assays, only one PCR strand is captured on the surface after denaturation. However, due to re-annealing to the complementary strand and steric hindrance of the surface, the capture efficiency was extremely low, and had a significant effect on the assay sensitivity. Multiplexing detected mutations is even more challenging because it is difficult to design capture reporters that selectively bind their complementary PCR strands at a single temperature. Previously used "amplicon reduction"25Attempts to formally overcome this problem have been successful, allowing highly sensitive detection of single mutations. Unfortunately, several disadvantages prevent the use of assays with true clinical samples. In the "amplicon reduction" method, a number of different PCR products are spotted, subsequently denatured and hybridized to two oligonucleotides labeled with Cy3 and Cy5 that are complementary to both wild type and mutant sequences. In this way, samples from several patients can be genotyped in a single chip, but a different chip is required for each mutation. Furthermore, PCR products obtained in a clinical setting must be arrayed on a silicon slide, which makes the assay impractical and cumbersome.
In this study, a new series of manipulations was designed to give a high degree of multiplexing and robustness to the assay. The biotinylated forward primer allows the PCR products to bind to streptavidin-modified magnetic beads. Amplicons bound to magnetic beads were heat denatured (5 min at 95 ℃) and rapidly placed on a magnet. The supernatant with single-stranded DNA was recovered while the magnetic beads with biotinylated strands were discharged. The single stranded amplicons are hybridized in solution to oligonucleotide reporters. Removal of the unwanted DNA strand is important to improve assay sensitivity because it prevents re-annealing of the two strands prior to hybridization with the reporter.
For this embodiment, the sequence of steps after amplification is summarized in FIG. 2.
Single-stranded PCR was incubated with stabilizer oligonucleotides for 10 minutes to prevent secondary structure formation in the DNA, which would interfere with hybridization to a specific reporter. For some mutations, this step is an important part of the process, as in the absence of a stabilizer, the assay does not work in some cases (data not shown).
The specificity of the assays described herein depends largely on allele-specific in-tube hybridization (10 min) of the dual-domain reporter to the single-stranded amplicon containing the mutation site at a particular hybridization temperature. The sequences of the stabilizer and reporter oligonucleotides designed to detect the most common mutations in KRAS, BRAF, PIK3CA and NRAS are shown in tables 1, 2, 3 and 4, respectively. By performing hybridization in solution, the yield and specificity of molecular recognition is greatly improved. The key to successful genotyping by microarray technology is decoupling sequence recognition from surface capture. Solution hybridization allows identification with a thermal gradient so that each reporter oligonucleotide can bind to the amplicon under optimal conditions. Finally, a solution containing single-stranded PCR hybridized to the dual-domain reporter is contacted to the microarray surface. Oligonucleotides (barcode probes), 20-21 bases, are covalently bound at known positions on the surface of a silicon chip coated with a three-dimensional copolymer, known as a copolymehzed (DMA-NAS-MAPS). These barcode probes specifically hybridize to a two-domain reporter containing a sequence complementary to the barcode. The selection of the barcode probe sequence is important because it affects the efficiency and specificity of the detection. To minimize cross-hybridization with the dual domain oligonucleotide reporter and the universal oligonucleotide labeled with Cy3 (U-Cy 3), the barcode probe was tested experimentally for cross-hybridization (data not shown). Importantly, since the barcode probe is an artificial sequence, many mutations can be detected using the same barcode microarray simply by changing the portion of the two-domain reporter that is complementary to the mutated sequence.
To assess the reliability of this KRAS mutation test, genomic DNA extracted from subjects carrying different mutations in KRAS codons 12 (G12A, G12C, G12D, G12R, G12S, G12V) and 13 (G13D) and in wild-type controls was examined. As shown in fig. 3 and 5, this test successfully genotyped all 7 KRAS mutations correctly. The intense fluorescent signal only appears at the location where the oligonucleotide complementary to the barcode sequence is immobilized, and cross-hybridization is very low and has good reproducibility from point to point.
1.3. Detection limit
Clinical tumor samples (mainly from Formalin Fixed Paraffin Embedded (FFPE) tissues) usually consist of both wild type and mutant DNA, and the proportion of wild type DNA is usually far in excess of that of the mutant. The same is true for circulating tumor DNA (a typical marker in liquid biopsies). PCR sequencing methods are generally considered the gold standard for clinical diagnosis, but are reliable only when the mutant to wild-type ratio reaches 10% -20%31. Obtaining a homologous tumor sample in a clinical setting is very difficult, and therefore the mutation content can be below the detection limit of PCR sequencing. Therefore, there is an urgent need for more sensitive KRAS mutation detection methods to improve clinical diagnosis. The sensitivity of the system was assessed experimentally by serial dilution (5%; 2.5%; 1.25%; 0.62%; 0.31%; 0.075%; 0.037%) of mutant DNA appropriately mixed with wild-type DNA.
Specifically, the heterozygous reference standard (50% mutated G12D allele) of Diatech Pharmacogenetics was used as the starting point. The microarray system described herein was able to detect a minimum of about 0.075% of mutant alleles in a G12D mutant wild-type DNA background (fig. 4). This is a large, unexpected improvement in PCR sequencing sensitivity that can detect as few as 10% of mutants. This level of sensitivity is mandatory in the detection of mutations in cell-free circulating DNA in liquid biopsies.
1.4. KRAS mutations were detected in Formalin Fixed Paraffin Embedded (FFPE) cancer tissue.
The assay was validated by analysis of DNA extracted from FFPE clinical samples from subjects in which the KRAS gene, previously encoded by QX100, was mutated or wild-typeTMDroplet DigitalTMPCR (ddPCR) System (Bio-Rad). The results obtained are shown in fig. 5. The measurement can be made clearlyThe correct mutation was detected in each clinical sample analyzed.
Furthermore, to assess the applicability of the tag-microarray approach in a real clinical setting, we performed blind analysis on 15 FFPE samples. To correctly genotype tumor DNA with a single chip, single-stranded PCR was hybridized with a mixture of 7 specific reporters at a stepwise temperature gradient ranging from 41 ℃ (G12C mutation-specific hybridization temperature) to 29 ℃ (G12V mutation-specific hybridization temperature). The mixture was kept at 41, 37, 36 and 30 and 29 ℃ for 5 minutes, corresponding to the hybridization temperatures of the G12C, G12D, G13D, G12S, G12A, G12R and G12V mutations, respectively. All 15 samples were analyzed in parallel and correctly genotyped in less than 40 minutes. Typical results are shown in fig. 6 for the samples identified with the G12S mutation. The wild type was always present, while different 6X6 spots highlighted repeats at different positions on the slide depending on the mutations detected.
2. Experimental part
2.1. Sample (I)
15 Formalin Fixed Paraffin Embedded (FFPE) samples were analyzed using this procedure.
Circulating tumor DNA was extracted using the automatic extractor Maxwell (R) RSC ccfDNA plasmid Kit (catalog No. AS1480) with Maxwell RSC (Promega).
To evaluate the sensitivity of this method, a dilution curve was generated starting from a heterozygous reference standard by Diatech pharmacopoetics (50% mutated allele) mixed with wild-type DNA in a ratio that mimics the concentration of tumor DNA in the plasma of cancer patients.
PCR conditions
PCR was performed on DNA containing 15ng, 200. mu.M of each deoxynucleotide, 10mM Tris-HCl (pH 8.3), 50mM KCl, and 1.5mM MgCl21U DNA polymerase (FastStart; Roche) and 10 pmoles of each primerThis was done in 25. mu.L reaction. Cycling conditions required initial denaturation at 95 ℃ for 10 minutes, followed by 35 cycles at 95 ℃ for 30 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 30 seconds, and final extension at 72 ℃ for 10 minutes.
2.3. Silicon slide coating and microarray preparation
2.4. Preparation of Single stranded DNA from PCR products Using streptavidin magnetic beads and liquid allele specific hybridization
Streptavidin-coated magnetic beads (Dynabeads) were used prior to useTMM-270 Streptavidin, Invitrogen) was purified using binding and washing buffer (B & W) (5mM Tris-HCl, pH 7.5; 0.5mM EDTA; 1M NaCl) was washed three times according to the manufacturer's protocol. Streptavidin-coated beads (250. mu.g) were then added to PCR tubes containing 25. mu.L of biotinylated PCR product and 75. mu. L B & W buffer and incubated at room temperature for 10 min with gentle rotation. The beads with bound PCR products were then washed 2-3 times in B & W buffer and resuspended in 30. mu.L of the same buffer and heated to 95 ℃ for 5 minutes. At the end of the denaturation step, the PCR tube was placed on a magnet and the supernatant with single-stranded PCR (29.1 μ Ι _) was transferred to a new tube (final concentration 0.3 μ Ι _) containing 0.9 μ Ι _, 10 μ Μ stabilizer, which is the oligonucleotide necessary to open the secondary structure present in the amplicon, and incubated with this oligonucleotide for 10 min at room temperature while the beads with bound biotinylated PCR strands were drained. Then to detect wild-type and G12A, G12C, G12D, G12R, G12S, G12V and G13D KRAS mutations, the reporters for the wild-type and mutant sequences were added together in equimolar amounts (final concentration 0.1 μ M) to the tubes containing the ssPCR-stabilizer solution (final volume 40 μ L). Incubation was gently rotated for 35 minutes at a stepwise temperature gradient (where the steps correspond to hybridization temperatures specific for the seven mutations) (see table 1).
2.5. Microarray hybridization, image scanning and data analysis
After liquid allele-specific hybridization, universal oligonucleotides labeled with Cy3 (universal-Cy 3) were added to ssPCR-reporter solution to a concentration of 0.3 μ M in order to detect KRAS mutations. The solution was then spread on a spotting silicon chip and a cover slip was placed on the spotting area. The chip was incubated in a humidified hybridization chamber for 15 minutes at room temperature.
Finally, the silicon chip was removed from the hybridization chamber and briefly soaked in 4 XSSC buffer to remove the coverslip, washed twice in 2 XSSC/0.1% SDS at room temperature for 5 minutes, then sequentially soaked in solutions 0.2 XSSC and 0.1XSSC at room temperature for 1 minute, dried with a stream of nitrogen and scanned. The hybridization chip was scanned using a ProScanArray (Perkin Elmer, MA, USA). Specifically, green laser (λ) was applied to Cy3 dyeex543 nm/λem570 nm). Photomultiplier tube (PMT) tube gain and laser power were varied between different experiments. 16-bit TIFF images were analyzed at 5 μm resolution. Data intensity was extracted with a scanner (Scanarray Express) and data analysis was performed for each experiment.
Unexpected conclusions
In summary, in this preferred embodiment, a microarray platform for rapid, specific and sensitive detection of mutations in
Very unexpectedly, microarray-based assays achieved the level of sensitivity in detecting few mutations reported in this work. The most significant advantage of this system is the ability to separate mutation detection from array hybridization without the use of enzymatic reactions, such as ligases or single base extensions. Each step is improved individually, and thus the sensitivity and accuracy of the method is significantly improved. Direct hybridization DNA microarrays have differential hybridization efficiencies due to sequence variations or the amount of target present in the sample, and background noise and false signals are increased due to mismatched hybridization and non-specific binding. Instead, this method can easily distinguish all point mutations in solution, and then allow rapid hybridization of the array at room temperature using divergent surface barcode probe sequences with similar properties. Using the tag-microarray approach, genotyping of clinical samples can be achieved in less than 90 minutes, thus, the assay greatly reduces operating time, for example, compared to direct sequencing methods which typically require 1-2 working days.
Finally, the proposed method can be a universal method, as the spotted barcode probe sequences remain constant and their complements can be appended to any reporter pool that recognizes different codons or multiple mutations in different genes. Thus, a single array design can be programmed to detect a wide range of gene mutations. This method is an innovative technique and can be used not only for the conventional diagnosis of KRAS mutations but also for a wide range of genetic variations including, for example, BRAF, PIK3CA and NRAS mutations.
The new working example:
to further validate the proposed mutation assay, genomic DNA extracted from subjects carrying different mutations in: KRAS codons 12 (G12A, G12C, G12D, G12R, G12S, G12V), 13 (G13D), 61 (Q61 HC, Q61HT, Q61K, Q61L, Q61R) and 146 (a 146T), NRAS codons 12 (G12A, G12C, G12D, G12S, G12V) and 13 (G13D, G13R, G13V), and BRAF codon 15 (V600E) as well as wild-type controls. FIG. 12 shows the spotting protocol of the array and some examples of genotyping of KRAS (G12S, Q61R, A146T), NRAS (G13R) and BRAF (V600E) mutations. All considered KRAS, NRAS and BRAF mutations were found to be correctly genotyped. The intense fluorescent signal only appears at the oligonucleotide-fixed positions complementary to the barcode sequence, and cross-hybridization is very low and has good signal reproducibility from point to point.
FIG. 12 shows (A) a schematic of a spotted barcode probe array. A silicon chip coated with copolymehzed (DMA-NAS-MAPS) and 10% NAS was used as a substrate for covalently attaching amino-modified barcode probe oligonucleotides spotted at discrete locations. Each position in the grid identifies a separate barcode probe address corresponding to KRAS codon 12-13 (yellow square), KRAS codon 61 (orange square), KRAS codon 146 (blue square), NRAS codon 12-13 (green square) and BRAF mutation (red square). The light grey part of the array was spotted with amino-modified oligonucleotides (COCU 8), gene-independent, used as reference points.
(B) The microarray scanned the Cy3 fluorescence signals from five different silicon chips. Each robotically spotted array was hybridized to a single-stranded PCR incubated with a double-domain reporter that directed the construct to the correct address. Fluorescence detection was obtained by incubating the array with a mixture of universal Cy 3-labeled oligonucleotides complementary to the tagged reverse primer of single-stranded PCR and with Cy 3-labeled oligonucleotides complementary to COCU8 (COCU 10). KRASG12S, KRASQ61R, KRASA146T, NRASG13R and BRAF V600E correspond to control samples containing the indicated mutations. All five samples of known genotypes (homozygous or heterozygous mutants in the case of BRAF V600E) were correctly identified.
Lowest limit of detection LOD:
clinical tumor samples, mainly from Formalin Fixed Paraffin Embedded (FFPE) tissue, are usually composed of wild type and mutant DNA, and the proportion of wild type DNA is usually far in excess of that of the mutant. Obtaining a homologous tumor sample in a clinical sample can be difficult, and thus the mutated DNA content can be below the detection limit for PCR sequencing. Analysis of circulating tumor dna (ctdna) is more challenging due to its low mutant allele frequency and large dynamic range. The level of ctDNA in cancer patients ranges from < 0.1% to > 50% of the total cfDNA. Therefore, the technical sensitivity and dynamic range of the assay are important for introducing ctDNA assays in the clinic.
The sensitivity of the system was assessed with serially diluted (2.5%; 1.25%; 0.62%; 0.31%; 0.15%; 0.075%) mutant DNA appropriately mixed with wild-type DNA, and the total DNA amount was 20 ng. A calibration curve was established for each of the seven most common mutations of the KRAS gene to calculate the lowest limit of detection (LOD) intended as the smallest mutation abundance that can be detected deterministically by assay. The probe array of fig. 12 was spotted into 7 wells on a coated silicon slide. Using a 16-well incubation chamber (Nexterion IC-16, SCHOTT), mixtures of wild type and mutant DNA at different ratios of 0-2.5% were incubated in parallel on different wells of the same slide (FIG. 13A). The control sample (0% mutant DNA) showed only a low fluorescence background signal. By plotting the percentage of signal fluorescence compared to mutant DNA, a calibration curve can be drawn, one for each mutation, from which the LOD value is extrapolated. Figure 13C shows an example of a calibration curve for the KRAS G12S mutation.The table (fig. 13D) reports the detection limit for each mutation. The determination of the LOD is based on the following equation: 3,3 sigma/s whereinsIs the slope of the calibration curve and σ is the standard deviation of the fluorescence background of the spots for the wild type sample. The sensitivity of the microarray system presented here ranged from 0.03% of the KRAS G12C and G12D mutations to 0.28% of the KRAS G13D mutation.
Figure 13 (A) picture shows the implementation of KRAS codon 12-13 mutation calibration curve settings. (B) Microarray scanning of the Cy3 signal and magnification of a portion thereof from the coated silicon slides showed the results of hybridization of four different concentrations of mutant DNA in four different wells. (C) The graph shows the relative fluorescence intensity of the signal corresponding to the Cy 3-labeled mutant single-stranded PCR bound to the G12S barcode probe. The points calculated as the average of the intensities of the four spots correspond to the percentage of KRAS G12S mutation in the KRAS wild type DNA background. The value at
Detection of KRAS mutations in Formalin Fixed Paraffin Embedded (FFPE) cancer tissue and cell free circulating tumor dna (ctdna):
to evaluate the applicability of the tag-microarray approach in a real clinical setting, 18 FFPE samples were analyzed. The codons of the gene and the mutation are known, but not the mutation itself. To correctly genotype tumor DNA with a single chip, single-stranded PCR was hybridized with a mixture of all specific reporters (22 mutant reporters and 5 wt reporters) at a stepwise gradient temperature ranging from 42 ℃ to 29 ℃. All 18 samples were analyzed in parallel and correctly genotyped in less than 90 minutes. Typical results are shown in figure 14 for samples identified with KRAS G12S (a), KRAS Q61HC (B) and NRAS G12V (C) mutations. The corresponding wild type was always present, while different 2X2 spots highlighted repeats at different positions on the slide depending on the mutation detected.
Fig. 14 shows the same spotting protocol for barcode sequences as fig. 12A. (A) Cy3 fluorescence image and relative fluorescence intensity plot of samples identified with KRAS G12S mutation. (B) Cy3 fluorescence image and relative fluorescence intensity plot of samples identified with KRAS Q61HC mutation. (C) Cy3 fluorescence image and relative fluorescence intensity plot of samples identified with NRAS G12V mutation. The bar is the average of the intensities of 4 spots (2 × 2 sub-arrays) per barcode probe sub-array. Error bars are the standard deviation of fluorescence intensity for each sample.
To evaluate the applicability of the tag-microarray approach in a practical clinical setting, 3 liquid biopsy samples were analyzed using circulating tumor dna (ctdna). Figure 15 shows a typical fluorescence image of an array for detecting one of the 22 mutations reported in the protocol of figure 12. ctDNA, 2 mutations and 1 wild type were analyzed in 3 patients. Although there is always a wild type signal present (upper left), only mutated DNA produces a signal in the square. From the intensity of these signals, the concentration of the mutant DNA can be estimated by a calibration curve.
Core/dendrimer embodiments and working examples
As described above, also provided is a composition comprising at least one reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of the oligonucleotide is for hybridizing to a single stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to a surface of at least one microarray probe, and further comprising a core surface functionalized with the first and second domains.
In particular, further preferred embodiments are provided which relate to polymers, dendrimers and/or dendrimers. See, for example, FIGS. 7-11. Dendrimers and dendrimers are known in the art. See, for example,Dendrimers and Other Dendritic Polymersj.m.j. Frechet and d.a. Tomalia, edit 2001; see also U.S. patent nos. 7,138,121; 5,175,270, respectively; and 6,806,047. As used herein, a dendrimer may be a highly branched structure. Branch branchThe polymer may be a molecule that contains a branched core structure inside, the core structure having a surface surrounding the core, and the surface may be reactive and may be functionalized. Functionally useful groups including oligonucleotides may be covalently attached to the surface. Multiple types of groups can be attached, and the ratio of the amounts of the different groups can be controlled to provide useful or optimal results. Different types of dendrimers can be prepared, but the preferred example is the azido dendrimer. See, for example, PCT/
In this embodiment, the reporter molecule of the hybridizing step may further comprise at least one dendrimer covalently bound to the first domain and the second domain. Thus, the first domain of an oligonucleotide used for hybridization to a single-stranded amplification product may be attached to a dendrimer surface, and the second domain of an oligonucleotide used for hybridization to a surface of at least one microarray probe may also be attached to the same dendrimer surface.
The ratio of the first domain to the second domain may be, for example, 20: 1 to 1:20, or 10: 1 to 1: 10. 10: 1 to 1: 1. or 5: 1 to 3: 1. or about 4: 1.
in another aspect, there is provided a method for determining a target gene fragment, comprising:
amplifying at least one target gene fragment comprising or potentially comprising at least one SNP region of interest to form an initial amplification product, wherein the fragment to be amplified is primed in the amplification to enable (1) separation of one single strand of the initial amplification product from another single strand in a subsequent step of the assay, and (2) detection of the separated single strands in a subsequent step of the assay;
separating at least one single-stranded amplification product from the initial amplification product, wherein the single-stranded amplification product comprises or may comprise at least one SNP region of interest;
hybridizing the single-stranded amplification product with at least one reporter molecule in solution, said reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of the oligonucleotide is for hybridizing to the single-stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the surface of the microarray,
wherein the reporter molecule comprises a core surface functionalized with a first domain and a second domain.
In another aspect, there is provided a method for determining a target gene fragment, comprising:
amplifying at least one target gene fragment comprising or potentially comprising at least one SNP region of interest to form an initial amplification product, wherein the fragment to be amplified is primed in the amplification to enable (1) separation of one single strand of the initial amplification product from another single strand in a subsequent step of the assay, and (2) detection of the separated single strands in a subsequent step of the assay;
separating at least one single-stranded amplification product from the initial amplification product, wherein the single-stranded amplification product comprises or may comprise at least one SNP region of interest;
hybridizing the single-stranded amplification product with at least one reporter molecule in solution, said reporter molecule comprising at least two different domains of an oligonucleotide, wherein a first domain of the oligonucleotide is for hybridizing to the single-stranded amplification product, and wherein a second domain of the oligonucleotide is for hybridizing to at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow hybridization;
contacting a solution of hybridized single-stranded amplification products with at least one microarray probe surface comprising at least one capture probe to allow hybridization;
detecting the presence of the hybridized single stranded amplification product on the surface of the microarray,
wherein the reporter is a composition as described and/or claimed herein.
In some embodiments, the core is a polymeric core. In some embodiments, the core is nucleotide-free. In some embodiments, the core is a water-soluble polymer core. In some embodiments, the core is a dendrimer core. In some embodiments, the core is a dendrimer core. In some embodiments, the core is covalently bound to the first domain and the second domain. In some embodiments, the core is an azido dendrimer core that is covalently bound to the first domain and the second domain.
In some embodiments, the core is a dendritic polymer core that is covalently bound to a first domain and a second domain, and the ratio of the first domain to the second domain is about 10: 1 to 1: 1.
in some embodiments, the compositions are suitable for use in microarray assays for genotyping a gene segment of interest, as described above, including the working examples above. In some embodiments, the composition is suitable for detecting single mutations of an oncogene. In some embodiments, the compositions are suitable for detecting at least one single mutation in the KRAS, NRAS, BRAF and/or PIK3CA oncogenes. In some embodiments, the first domain is used to detect at least one single mutation in the KRAS oncogene. In some embodiments, the first domain is used to detect at least one KRAS oncogene mutation at
Other embodiments provide assays comprising a step of hybridizing using a reporter molecule described and/or claimed herein.
Other embodiments provide methods of making a reporter as described and/or claimed herein, comprising surface functionalizing a core with a first domain and a second domain.
Other embodiments provide kits comprising a reporter as described and/or claimed herein.
Additional working examples
Additional non-limiting working embodiments are described below.
Material
2, 2-bis (hydroxymethyl) propionic acid-azide dendrimer trimethylolpropane core, 3 rd generation from Sigma-Aldrich. The dendrimer has 24 azide surface groups.
Oligonucleotides Wt and Mut were purchased from Metabion International AG (Steinkirchen, Germany) and have the following sequences:
spotting Probe Wt: NH25’-ACTCCAGTGCCAAGTACGAT-3’
Spotting probe Mut: NH25’-GGCTCACGTCTTATTTGGGC-3’
Oligo 4 complementary to PCR Mut: 5'-GCTGATGGCGT-3' DBCO
general-Cy 3 (complementary to the reverse tag primer of PCR): 5'-CTCAATGTTCGGACTCAG-3' are provided.
Method of producing a composite material
Preparation of Single stranded DNA from PCR products Using streptavidin magnetic beads and liquid allele specific hybridization
Streptavidin-coated magnetic beads (Dynabeads) were used prior to useTMM-270 Streptavidin, Invitrogen) with binding and washing buffer (B)&W) (5mM Tris-HCl, pH 7.5; 0.5mM EDTA; 1M NaCl) were washed three times according to the manufacturer's protocol. Streptavidin-coated beads (250. mu.g) were then added to PCR tubes containing 25. mu.L of biotinylated PCR product and 75. mu. L B & W buffer and incubated at room temperature for 10 min with gentle rotation. The beads with bound PCR products were then washed 2-3 times in B & W buffer and resuspended in 30. mu.L of the sameAnd heated to 95 ℃ for 5 minutes. At the end of the denaturation step, the PCR tube was placed on a magnet, the supernatant with single-stranded PCR (29.1. mu.L) was transferred to a new tube containing 0.9. mu.L of 10. mu.M stabilizer (sequence: 5'-gcaagagtgccttgacgatacagctattcag-3'), the oligonucleotide (final concentration 0.3. mu.M) necessary to open the secondary structure present in the amplicon, and incubated with this oligonucleotide for 10 minutes at room temperature while the beads with bound biotinylated PCR strands were discharged.
Conjugation of oligonucleotides to azido dendrimers
The oligonucleotides were set at 4: 1 in combination with the dendritic polymer: the oligonucleotides complementary to the PCR are 4 times as many as the oligonucleotides complementary to the deposition probe. The azide dendrimer stock solution in DMSO was diluted to 42 μ M in TBS buffer (0.5 mM tris-HCl pH 7.50.05 mM EDTA 0.1M NaCl); then 0.5 μ L of this solution was added to 4: 1 mixtures were all 100. mu.M in water (4. mu.L and 1. mu.L, respectively) and incubated overnight at 37 ℃ (Wt dendrimer). Similarly,
Then mixing the two solutions of the wild dendritic polymer and the mutant dendritic polymer to obtain a single solution containing the Wt dendritic polymer and the Mut dendritic polymer; then 0.4. mu.L of this solution was added to 40. mu.L of wild type or mutant single stranded PCR (prepared as described above) together with 0.3. mu.M of general-
Silicon slide coating and microarray preparation
The 5' terminal amino modified capture probes (spotting probe Wt and spotting probe Mut) from Metabion International AG (Steinkirchen, Germany) were dissolved at a concentration of 10. mu.M in a printing buffer (150 mM sodium phosphate pH8.5, 0.01% sucrose monolaurate) and printed in 64 replicates (8X 8 subarrays) using a piezo spotter SciFlex ARRAYER S5 (science, Berlin, Germany). Spotting was performed in an atmosphere of 60% humidity at 20 ℃. After the spotting step, the chips were placed in an uncovered storage box, placed in a sealed chamber, and plated with sodium chloride (40 g/100 mLH)2O) and incubated overnight. After incubation, the chips were immersed in a preheated blocking solution (50 mM ethanolamine, 0.1M Tris, pH 9.0) at 50 ℃ for 15 minutes, followed by distillation H2Rinse twice in O to block all residual reactive groups of the coating polymer. The chips were washed in preheated post-coupling wash solution 4X sodium citrate brine (SSC), 0.1% (w/v) Sodium Dodecyl Sulfate (SDS) at 50 ℃ for 15 minutes with distilled H2Rinsed with O and dried by a stream of nitrogen.
Each chip was then incubated in a humidified chamber at room temperature for 1 hour with 15uL of a dendrimer solution hybridized with Wt or Mut PCR. Specifically, chip A was incubated with Wt PCR-modified dendrimer, and chip B was incubated with Mut PCR-modified dendrimer.
After incubation, the chip was removed from the hybridization chamber and briefly soaked in 4 XSSC buffer to remove the coverslip, washed twice in 2 XSSC/0.1% SDS for 5 minutes at room temperature, then sequentially soaked in solutions 0.2 XSSC and 0.1XSSC for 1 minute at room temperature, dried with a nitrogen stream and scanned. The hybridization chip was scanned using a ProScanArray (Perkin Elmer, MA, USA). Specifically, green laser (λ) was applied to Cy3 dyeex543 nm/λem570 nm). The photomultiplier tube (PMT) tube gain was set to 80 and the laser power was set to 90. 16-data intensities were extracted with a scanner (Scanarray Express) and data analysis was performed for each experiment.
Results
In this preferred embodiment, each dendrimer is modified with two oligonucleotides: one complementary to the probe immobilized on the silicon chip and the other complementary to the PCR sequence, as shown in FIG. 10.
In this preferred embodiment, the optimal ratio between the two oligonucleotides is 4: 1, in particular 4 times as many oligonucleotides complementary to the PCR as to the deposition probe.
As shown in FIG. 11A, each chip was spotted with Wt and mutant probes; thus, each chip was hybridized with a solution containing Wt dendrimer and Mut dendrimer. The fluorescence image (fig. 11B) shows that Wt DNA is highly specific captured to the surface by the barcode sequence. Mut PCR is captured by its dendrimers in solution and on the surface by its barcode sequence. The specificity in this case is not 100%, since a small number of dendrimers functionalized with oligonucleotides complementary to the mutated sequence are captured by the wild-type barcode sequence. Since the main objective is to assess the presence of mutated DNA, this type of cross-talk does not impair the quality of the assay. The fluorescence signal intensity relative to wild type and mutated captured dendrimers is reported in figure 11.
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Additional description
A rapid and sensitive microarray-based assay for detecting single mutations in, for example, KRAS oncogenes is described. In one third of human cancers, there are KRAS mutations that play a key role in the early development of cancer and resistance to standard treatment regimens. KRAS point mutations are clustered in several hot spots, mainly involving
