阅读说明：本技术 用于检测样本中的核酸的数位聚合酶连锁反应方法 (Digital polymerase chain reaction method for detecting nucleic acid in sample ) 是由 邱国平 于 2019-10-09 设计创作，主要内容包括：本发明涉及一种检测样本中核酸(NA)分子的方法。更具体而言,本发明涉及一种用于检测特定核酸序列的改良的基于数位PCR的方法。本发明可用于研究及诊断应用,具提高的灵敏度及准确性。本发明还提供一种试剂盒,用于执行本文所述的评估样本中核酸的方法。(The present invention relates to a method for detecting Nucleic Acid (NA) molecules in a sample. More specifically, the present invention relates to an improved digital PCR-based method for detecting specific nucleic acid sequences. The invention can be used for research and diagnosis application, and has improved sensitivity and accuracy. The invention also provides a kit for performing the methods of assessing nucleic acid in a sample described herein.)

1. A method of analyzing nucleic acids in a sample, the sample comprising one or more linear, double-stranded nucleic acid fragments (NA fragments), the method comprising the steps of:

(a) subjecting the sample to a 3' -a tail processing reaction that allows addition of adenine nucleotide (a) at the 3' -tail of the NA fragment to produce a 3' -adenine nucleotide (3' -a) overhang nucleic acid fragment (3' -a overhanging NA fragment);

(b) providing a double-stranded homogeneous adaptor of a 3' -thymine (T) or 3' -uracil (U) nucleotide overhang comprising a P oligonucleotide strand with a 5' -phosphate to join to the NA segment of the 3' -A overhang and a T/U oligonucleotide strand with a 3' -T or 3' -U without a 5' -phosphate, wherein the T/U oligonucleotide strand is complementary to the P oligonucleotide strand except at the 3' -T or 3' -U of the T/U oligonucleotide strand;

(c) subjecting the sample of (a) to a ligation reaction to ligate the homogeneous adaptor to the 3' -a overhanging NA fragment at both ends to generate adaptor-ligated nucleic acid fragments (adaptor-ligated NA fragments);

(d) combining the sample of (c) with Polymerase Chain Reaction (PCR) reagents and detection reagents to provide a sample ready for amplification/detection, wherein the PCR reagents comprise a single primer for amplification having the nucleic acid sequence of the T/U oligonucleotide strand and the detection reagents comprise one or more fluorescent probes that generate a fluorescent signal and specifically hybridize to the NA fragments;

(e) dividing the amplification/detection-ready sample of (d) into a plurality of partitions, each partition containing a limited number of copies of the adaptor-ligated NA fragments;

(f) performing PCR in each partition using the adaptor-ligated NA fragment as a template and the singleton primer as a forward and reverse primer to amplify the adaptor-ligated NA fragment; and

(g) the fluorescence signal of each fraction was evaluated.

2. The method of claim 1, wherein step (g) comprises detecting the fluorescence color (signal) of all partitions containing amplified adaptor-ligated NA fragments and counting the number of partitions with the expected fluorescence color, thereby determining the estimated amount of NA fragments in the sample.

3. The method of claim 1, wherein in step (e) more than 50% of the partitions contain no more than one duplicated adaptor-ligated NA fragment.

4. The method of claim 1, wherein in step (e), each partition comprises at least one duplicate of the adaptor-ligated NA fragments.

5. The method of claim 1, wherein the NA segment comprises a nucleic acid sequence indicative of the health/disease state of the subject.

6. The method of claim 1, wherein the homogeneous adaptors of step (b) are not self-ligated.

7. The method of claim 1, wherein the homogeneous adaptor of step (b) has a 3' -T or 3' -U overhang in the T/U oligonucleotide strand and a 3' -non-a overhang in the P oligonucleotide strand.

8. The method of claim 1, wherein the homogeneous adapter of step (b) has a 3' -T overhang at one end and a blunt end at the other end.

9. The method of claim 1, wherein the sample is obtained from a body fluid sample.

10. The method of claim 1, wherein the NA segments in the sample are cell-free DNAs.

11. The method of claim 1, further comprising performing a terminal repair reaction on the NA segment prior to step (a).

12. The method of claim 1, wherein the PCR of step (f) is performed by oil emulsion or droplet PCR, or well-based PCR.

13. The method of claim 1, wherein the determining of step (g) is performed by flow cytometry using a fluorescent probe.

14. The method of claim 1, further comprising performing reverse transcription-PCR (RT-PCR) on the sample to convert the RNA into linear, double-stranded complementary DNA (cDNA) prior to step (a).

15. A kit for performing a method of measuring the amount of nucleic acid fragments in a sample comprising

(i) An adaptor ligation reagent comprising a homogeneous adaptor, a ligation buffer, and a ligase, wherein the adaptor comprises a P oligonucleotide strand with a 5 '-phosphate and a T/U oligonucleotide strand with a 3' -T or 3-U and no 5 '-phosphate, wherein the T/U oligonucleotide strand is complementary to the P oligonucleotide strand, except at the 3' -T or 3'-U of the T/U oligonucleotide strand, wherein the homogeneous adaptor is capable of ligating to the nucleic acid fragments at both ends, wherein the nucleic acid fragments have 3' -A overhangs;

(ii) PCR reagents comprising a single primer as the only primer, having the nucleic acid sequence of the T/U oligonucleotide strand, dNTPs, PCR buffer, and DNA polymerase; and

(iii) a detection reagent comprising one or more detectable probes having a complementary sequence that specifically hybridizes to the nucleic acid fragment.

16. The kit of claim 15, further comprising instructions for use, wherein the instructions for use comprise instructions for performing a method comprising the steps of:

(a) subjecting the sample to a 3' -a tail processing reaction that allows addition of adenine nucleotide (a) at the 3' -tail of the NA fragment to produce a 3' -adenine nucleotide (3' -a) overhang nucleic acid fragment (3' -a overhanging NA fragment);

(b) providing a double-stranded homogeneous adaptor of a 3' -thymine (T) or 3' -uracil (U) nucleotide overhang comprising a P oligonucleotide strand bearing a 5' -phosphate to join to the NA segment of the 3' -a overhang and a T/U oligonucleotide strand bearing a 3' -T or 3' -U without a 5' -phosphate, wherein the T/U oligonucleotide strand is complementary to the P oligonucleotide strand except at the 3' -T or 3' -U of the T/U oligonucleotide strand;

(c) subjecting the sample of (a) to a ligation reaction to ligate the homogeneous adaptor at both ends to the 3' -a overhanging NA fragment to generate adaptor-ligated nucleic acid fragments (adaptor-ligated NA fragments);

(d) combining the sample of (c) with Polymerase Chain Reaction (PCR) reagents and detection reagents to provide a sample ready for amplification/detection, wherein the PCR reagents comprise a single type primer for amplification having the nucleic acid sequence of the T/U oligonucleotide strand and detection reagents for detection comprising one or more fluorescent probes that generate a fluorescent signal and specifically hybridize to the NA fragment;

(e) dividing the amplification/detection-ready sample of (d) into a plurality of partitions, each partition containing a limited number of copies of the adaptor-ligated NA fragments;

(f) performing PCR in each partition using the adaptor-ligated NA fragment as a template and the singleton primer as a forward and reverse primer to amplify the adaptor-ligated NA fragment; and

(g) the fluorescence signal of each fraction was evaluated.

Technical Field

The invention relates to a method for detecting Nucleic Acid (NA) molecules in a sample. More specifically, the present invention relates to an improved digital PCR-based method for analyzing nucleic acid fragments. The invention can be used for research and diagnosis application, and has improved sensitivity and accuracy. The invention also provides a kit for performing the method for detecting nucleic acid in a sample as described herein.

Background

Various techniques for nucleic acid detection have been developed for a number of research and diagnostic applications. Digital polymerase chain reaction (dPCR) is considered to be one of the most precise quantitative methods for analyzing gene Copy Number Variations (CNVs), gene expression, genetic mutations, and single-nucleotide polymorphisms (SNPs). Its popularity is constantly increasing. Many companies have designed company specific experimental methods, hardware and software applications (Dong et al, 2015; Morley, 2014; Zhao et al, 2016).

The most common model is droplet-based and titer plate-based digital PCR methods. Currently, droplet-based digital PCR is sold by Bio-Rad. The QX200 Droplet Digital PCR (ddPCR) instrument is a recently marketed product by Bio-Rad, one of the most advanced models to divide PCR reactions into water-in-oil droplets for absolute nucleic acid quantification combining microfluidic and surfactant chemistry techniques (Hindson et al, 2011). This method allows analysis of droplets of approximately 20,000 nanoliters per sample run, making it the most efficient method in a similar instrument. On the other hand, the Qiagen company (http:// www.captodayonline.com/high-throughput-digital-PCR-system-1017/) was used as an example to perform the reaction on the titration plate based on digital PCR. For this method, DNA molecules are diluted and dispensed into, for example, 96-well titration plates for independent PCR reactions. After amplification, all wells were probed and quantified by gene-specific sequences to identify wells with positive reactions.

As a PCR-based nucleic acid detection technique, digital PCR typically requires amplification of paired primers and detection of target nucleic acids using probes, dilution and distribution of the sample in order to separate nucleic acid fragments therein for separate reactions, each with a very limited number of target nucleic acid molecules. In this way, PCR reactions can be performed separately within each partition and the signal in each partition determined to be negative or positive, and thus the exact number of copies of the nucleic acid sequence in the original sample can be determined by counting the number of positive partitions (sequences detected) and negative partitions (sequences not detected) based on Poisson distribution. In general, the experimental procedure of digital PCR includes the following steps: 1) diluting the target DNA molecule; 2) dividing the well-separated target DNA fragments into discrete droplets/chambers, each droplet/chamber further comprising other components required for amplification and signal detection; 3) PCR amplification using "paired (paired)" gene specific primers for the "internal (internal)" region of the target gene; 4) detecting a fluorescent signal using a fluorescent probe; 5) quantifying positive and negative reactions based on poisson distribution; and 6) cross-sample comparison to determine significance.

Cell-free nucleic acid samples are readily available, non-invasive genetic material, and are becoming increasingly popular in diagnosing a variety of diseases (Wagner, 2012). These genetic materials are released from all cells in the body, including normal cells, diseased cells, and microorganisms. Theoretically, cell-free nucleic acids may be present in a variety of bodily fluids, including blood, saliva, urine, leucorrhea, semen, lymph, and sweat (Chiu and Yu, 2019; Nai et al, 2017; Wagner, 2012). Despite many advantages, the number of cell-free nucleic acid samples is often small, making these samples difficult to handle and therefore easily lost during the course of the experiment. Furthermore, cell-free nucleic acids are also highly fragmented. These all show particularly serious problems with precious materials.

Current digital PCR still faces many challenges in cycling cell-free nucleic acid analysis. Several methods have been proposed to improve current digital PCR, such as appropriate preservation of nucleic acid samples to reduce degradation of cell-free DNA (cfDNAs), efficient purification such as silica gel membrane to collect sufficient amount of cfDNAs for detection, and increase detection sensitivity by high-sensitivity capillary electrophoresis. Current digital PCR also has the disadvantage that probes are designed based on specific known mutation sites to detect certain mutations in nucleic acids, but it does not cover all genetic variations, thus limiting the testing of other potential variations or mutations.

Therefore, there is a need to develop improved digital PCR methods for nucleic acid detection.

Disclosure of Invention

The present invention provides an improved digital PCR for detection of nucleic acids in a sample, referred to as digital T-oligo PCR (digital TOP-PCR). The digital TOP-PCR of the invention is characterized by the use of Homogeneous Adaptors (HA) ligated to the ends of all nucleic acid fragments in the sample and unique primers (T/U oligonucleotides) that recognize complementary sequences in HA and serve as forward and reverse primers for amplification. The digital TOP-PCR of the present invention can amplify all nucleic acid fragments in a sample, and further can achieve the subsequent detection of target nucleic acid by increased sensitivity, especially by reducing the false negative rate.

In particular, the present invention provides a method for analyzing nucleic acids in a sample comprising one or more linear, double-stranded nucleic acid fragments (NA fragments), the method comprising the steps of:

(a) subjecting the sample to a 3' -a tail processing reaction that allows addition of adenine nucleotide (a) at the 3' -tail of the NA fragment to produce a 3' -adenine nucleotide (3' -a) overhang nucleic acid fragment (3' -a overhanging NA fragment);

(b) providing a double-stranded homogeneous adaptor of a 3' -thymine (T) or 3' -uracil (U) nucleotide overhang comprising a P oligonucleotide strand bearing a 5' -phosphate to join to the NA segment of the 3' -a overhang and a T/U oligonucleotide strand bearing a 3' -T or 3' -U without a 5' -phosphate, wherein the T/U oligonucleotide strand is complementary to the P oligonucleotide strand except for the 3' -T or 3' -U in the T/U oligonucleotide strand;

(c) subjecting the sample of (a) to a ligation reaction to ligate the homogeneous adaptor to the 3' -a overhanging NA fragment at both ends to generate adaptor-ligated nucleic acid fragments (adaptor-ligated NA fragments);

(d) combining the sample of (c) with Polymerase Chain Reaction (PCR) reagents and detection reagents to provide a sample ready for amplification/detection, wherein the PCR reagents comprise a single type primer for amplification having the nucleic acid sequence of the T/U oligonucleotide strand and the detection reagents comprise one or more fluorescent probes for detection that generate a fluorescent signal and specifically hybridize to the NA fragment;

(e) dividing the amplification/detection-ready sample of (d) into a plurality of partitions, each partition containing a limited number of copies of the adaptor-ligated NA fragment;

(f) performing PCR in each partition using the adaptor-ligated NA fragment as a template and the singleton primer as a forward and reverse primer to amplify the adaptor-ligated NA fragment; and

(g) the fluorescence signal of each fraction is evaluated.

In some embodiments, step (g) comprises determining whether the droplets/fractions are positive or negative based on the intensity of the fluorescent signal, and then calculating the total number (counts) of droplets/fractions having a positive signal.

In some embodiments, in step (e), more than 50% of the partitions contain no more than one copy of the adaptor-ligated NA fragments.

In some embodiments, in step (e), each partition comprises at least one duplicate of the adaptor-ligated NA fragments.

In some embodiments, the NA fragment comprises a nucleic acid sequence that is indicative of the health/disease state of the individual.

In some embodiments, the homogeneous adaptors of step (b) are not self-ligated.

In some embodiments, the homogeneous adapter of step (b) has a 3' -T or 3' U overhang in its T/U oligonucleotide strand and a 3' -non-A overhang in its P oligonucleotide strand.

In some embodiments, the homogeneous adapter of step (b) has a 3' -T overhang at one end and a blunt end at the other end.

In some embodiments, the sample is obtained from a bodily fluid, including, but not limited to, blood, urine, saliva, tears, sweat, breast milk, nasal secretions, amniotic fluid, semen, and vaginal fluid.

In some embodiments, the NA fragments in the sample are cell-free DNAs (cfDNAs).

In some embodiments, prior to step (a), the methods described herein further comprise performing a terminal repair reaction on the NA fragment.

In some embodiments, the PCR of step (f) is performed by oil emulsion or droplet PCR or well-based PCR.

In some embodiments, the determination of step (g) is performed by flow cytometry using a fluorescent probe.

In some embodiments, the method of the present invention comprises steps (a) through (g) and optionally step (a)': if the NA segment contains linear, single-stranded RNAs, the sample is subjected to reverse transcription-PCR (RT-PCR) prior to step (a) to convert the RNAs into linear, double-stranded complementary DNA (complementary DNA, cDNA).

The invention also provides a kit for performing the method of detecting nucleic acid fragments in a sample as described herein. In particular, the kit comprises

(i) An adaptor ligation reagent comprising a homogeneous adaptor, a ligation buffer, and a ligase, wherein the homogeneous adaptor comprises a P oligonucleotide strand with a 5 '-phosphate and a T/U oligonucleotide strand with a 3' -T or 3-U without a 5 '-phosphate, wherein the T/U oligonucleotide strand is complementary to the P oligonucleotide strand except at the 3' -T or 3'-U of the T/U oligonucleotide strand, wherein the homogeneous adaptor is capable of ligating to the nucleic acid fragments at both ends, wherein the nucleic acid fragments have 3' -A overhangs;

(ii) PCR reagents comprising a single primer (the only primer) having the nucleic acid sequence of the T/U oligonucleotide strand, dNTPs, PCR buffer, and DNA polymerase; and

(iii) a detection reagent comprising one or more detectable probes having a complementary sequence that specifically hybridizes to the nucleic acid fragment.

In some embodiments, the kit further comprises instructions for use, wherein the instructions for use comprise instructions for performing a method comprising steps (a) to (g) described herein.

The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following detailed description of several specific embodiments, and from the claims.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

FIG. 1 shows the procedure of the method of the present invention. The ratio of the P oligonucleotide: 5'-GTCGGAGTCTgcgc-3' (SEQ ID NO: 24). T-oligonucleotide: 5 '-AGACTCCGAC(T) -3' (SEQ ID NO: 23).

FIG. 2 shows the difference between the method of the present invention and a conventional PCR-based detection method. The cfDNA sample contains a set of cfDNA fragments with random breaks from genomic sources. In conventional PCR-based detection methods, only the cfDNAs fragment "g" covering the first given primer binding site and the second given primer binding site can be amplified and detected. As a result, the sensitivity of the detection is limited, especially when the nucleic acid content is low, the sensitivity is even worse. In contrast, in the methods of the invention, all cfDNA fragments can be amplified uniformly and universally, and after such amplification, not only fragment "g" but also other fragments "a" to "f" (having only one primer binding site, or even no primer binding sites) are amplified, all from the same pathogenic nucleic acid of interest (same genomic origin) can be detected, e.g., using one or more probes carrying a detectable label capable of specifically hybridizing to any region within the pathogenic nucleic acid of interest; as a result, since amplification before detection is applied to all nucleic acid fragments as well (non-specifically), the sensitivity of detection is improved and false negatives can be minimized, and thus the relative amount of each amplified nucleic acid fragment can represent the relative amount present in the original sample.

FIG. 3 shows the counts of positive droplets generated from conventional ddPCR and ddTOP-PCR methods using samples containing different amounts of partial and complete template. Lanes 1 and 2(A05 vs A06) were generated using T/U oligonucleotide primers and N-myc gene specific primers, respectively, amplified from the NAGK gene sequence deleted from the 5 'primer binding site, with the remainder (lanes 3-16 or B05-H06) generated from a mixed template containing varying amounts (100% -0%) of portions (labeled "H", or 5' primer binding site deleted) and the entire template (labeled "F", or with two primer binding sites). All samples were tested using the same N-myc probe. The ddTOP-PCR count is marked "05" after the letter, while its corresponding ddPCR count is marked side by side with "06". The numbers in the figure represent counts (number of copies/microliter). A total of 20. mu.l of each sample was used for counting in the QX200ddPCR instrument. Ch1, channel 1, defined by QX 200.

FIG. 4 shows the comparison of fluorescence signal intensity between ddPCR and ddTOP-PCR. As in FIG. 3, lanes 1 and 2(A05 vs A06) were generated using T/U oligonucleotide primers and N-myc primers, respectively, amplified from a 5 'primer binding site deleted NAGK template, with the remainder (lanes 3-16 or B05-H06) generated from a mixed template containing varying amounts (100% -0%) of a portion (labeled "H", or 5' primer binding site deleted) and the entire template (labeled "F", or with two primer binding sites). All samples were detected using the same N-myc oligonucleotide probe. Note that a droplet displayed as a black dot is not considered a positive droplet because its intensity is below a preset threshold.

FIG. 5 shows the sequences listed in Table 1. T oligonucleotide: 5'-AGC GCT AGA CTC CGA CT-3' (SEQ ID NO: 1). P oligonucleotide, 5'-GT CGG AGT CTA GCG CT-3' (SEQ ID NO: 2).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the articles "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. For example, "a component" means one component or more than one component.

The terms "comprising," "including," "includes," "including," "contains," "containing," "involving," and the like are generally used in an inclusive/sense-referring to the permissible presence of one or more features, elements, or components. The words "comprising" or "including" and the like encompass "consisting of or" consisting of.

As used herein, "approximately," "about," or "approximately" may generally refer to within 20%, specifically within 10%, more specifically within 5% of a given value or range. Numerical values set forth herein are approximate, meaning that if not explicitly stated, the word "about", "approximately", or "approximately" may be inferred.

The terms "polynucleotide" or "nucleic acid" and the like refer to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs, including those having non-naturally occurring nucleotides. Polynucleotides can be synthesized using, for example, an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. It will be understood that when the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it also includes RNA sequences in which "T" is replaced with "U" (i.e., A, U, G, C). The term "oligonucleotide" refers to a relatively short nucleic acid fragment, typically less than or equal to 150 nucleotides in length, e.g., between 5 and 150. Oligonucleotides can be designed and synthesized as desired. In the case of primers, the length is generally from 5 to 50 nucleotides, in particular from 8 to 30 nucleotides. In the case of probes, they are generally 10 to 100 nucleotides, in particular 15 to 30 nucleotides, in length.

As used herein, the term "complementary" refers to the topological compatibility or matching together of the interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and in addition, the contact surface properties are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, a polynucleotide of sequence 5'-TATAC-3' is complementary to a polynucleotide of sequence 5 '-GTATA-3'.

As used herein, a target nucleic acid may refer to a particular target nucleic acid detected in a sample. Target nucleic acids include, but are not limited to, DNA, such as genomic DNA, mitochondrial DNA, cDNA, and the like, and RNA, such as mRNA, miRNA, and the like. The target nucleic acid may be derived from any source, including natural sources or synthetic sources. For example, the target nucleic acid may be from an animal or pathogen source, including, but not limited to, mammals such as humans, and pathogens such as bacteria, viruses, and fungi. The target nucleic acid can be obtained from any body fluid or tissue (e.g., blood, urine, skin, hair, feces, and mucus) or environmental sample (e.g., a water sample or a food sample). In some embodiments, the target nucleic acid can be a collection of nucleic acid molecules of the same origin (e.g., the same gene from a normal or diseased individual or pathogen), but of various lengths. For example, many fragments of the gene encoding hepatitis B surface antigen (HBsAg) are available as "target" nucleic acid fragments of various lengths present in the test sample. Since each target nucleic acid molecule comprises at least a portion of the HBsAg gene, probes or primers having sequences corresponding to (or complementary to) various positions within the HBsAg gene can be used to detect target nucleic acid fragments. As another example, the target nucleic acid can be a nucleic acid containing a genetic mutation (e.g., a Single Nucleotide Polymorphism (SNP) indicative of a disease such as cancer).

As used herein, the term "primer" refers to an oligonucleotide that can be used in an amplification method, e.g., Polymerase Chain Reaction (PCR), to amplify a target nucleotide sequence. In conventional PCR, at least one pair of primers, including a forward primer and a reverse primer, is required for amplification. In general, for a target DNA sequence to be amplified consisting of a (+) strand and a (-) strand, a forward primer is an oligonucleotide that hybridizes to the 3' end of the (-) strand, thereby initiating polymerization of a new (+) strand under reaction conditions; while the reverse primer is another oligonucleotide that hybridizes to the 3' end of the (+) strand under reaction conditions and thus can initiate polymerization of a new (-) strand under the reaction conditions. In particular, for example, the forward primer can have the same sequence as the 5 'end of the (+) strand, while the reverse primer can have the same sequence as the 5' end of the (-) strand. Typically, the sequences of the forward and reverse primers used to amplify the target nucleic acid sequence are different from each other. As used herein, a "single" primer refers to only one type of primer, all primers having the same sequence, rather than a pair of primers having different sequences, one primer being a forward primer and the other primer being a reverse primer.

As used herein, the term "hybridization" shall include any process by which a strand of nucleic acid joins with a complementary strand through base pairing. Related methods are well known in the art and are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, second edition, Cold spring harbor Laboratory Press (1989), and Frederick MA et al, Current Protocols in Molecular Biology, John Wiley&Sons corporation (2001). Generally, stringent conditions will be selected to give a thermal melting point (T) at a specified ionic strength and pH value in comparison to the specified sequence_m) About 5 to 30 deg.c lower. More typically, stringent conditions are selected to compare the T of the designated sequence at a defined ionic strength and pH_mAbout 5 to 15 deg.c lower. For example, stringent hybridization conditions will be those in which the salt concentration is less than about 1.0M sodium (or other salt-like) ion, typically about 0.01 to about 1M sodium ion concentration, at about pH 7.0 to about pH 8.3, and the temperature is at least about 25 ℃ for short probes (e.g., 10 to 50 nucleotides) and at least about 55 ℃ for long probes (e.g., greater than 50 nucleotides). Exemplary non-or low stringency conditions for long probes (e.g., greater than 50 nucleotides) will comprise 20mM Tris, pH 8.5, 50mM KCl, and 2mM MgCl₂The reaction temperature was 25 ℃.

As used herein, "overhang" refers to a fragment of a single unpaired nucleotide or a longer piece of unpaired nucleotide at the end of a linear double-stranded nucleic acid molecule. Unpaired nucleotides can be at the 3 'or 5' end, creating 3 'or 5' overhangs, respectively. A "3 '-A overhang" means that unpaired nucleotides are present at the 3' end and consist of one or more adenine (A) nucleotides. A "3 '-non-A overhang" means that unpaired nucleotides are present at the 3' end and do not include any adenine (A) nucleotides. A "3-T overhang" means that unpaired nucleotides are present at the 3' end and consist of one or more thymine (T) nucleotides.

"Single", "homologous" or "universal" primers refer to the presence of only one primer having the same sequence in a PCR reaction, rather than a pair of primers. The term "heterologous primers" refers to the presence of at least one pair of matched primers in a PCR reaction, each primer having a sequence different from the other.

As used herein, the term "adaptor" refers to an oligonucleotide that can be ligated to the end of a double-stranded nucleic acid molecule. The length of the adapter may be 10 to 50 bases, preferably 10 to 30 bases, more preferably 10 to 20 bases. A length of less than 10 nucleotides may reduce the specificity of annealing. Lengths in excess of 20 nucleotides may not be cost effective. The term "homogeneous" adaptor refers to a single type of adaptor used to ligate to both ends of a double stranded nucleic acid molecule. The term "heterologous" adaptor refers to at least two types of adaptors having nucleotide sequences that are different from each other, one for ligation to the 5 'end of the double stranded nucleic acid molecule and the other for ligation to the 3' end of the double stranded nucleic acid molecule.

To amplify nucleic acid fragments in a sample, we have developed a T oligonucleotide-based polymerase chain reaction (TOP-PCR) technique using a homogeneous adaptor composed of a P oligonucleotide and a T oligonucleotide ligated to the ends of all nucleic acid fragments, and then using the T oligonucleotide as a single primer to indiscriminately amplify all nucleic acid fragments in the sample. See U.S. patent No. 10,407,720, which is incorporated herein by reference in its entirety.

In the present invention, it was found that the conventional digital PCR can be significantly improved by using the TOP-PCR technique. By using TOP-PCR for amplification, the method of the invention is performed in a digital manner for nucleic acid detection, so that all nucleic acids in the reaction are amplified in equal proportion, and the target nucleic acid can be detected with higher sensitivity by one or more sequence-specific probes. As shown in the examples provided below, the methods of the present invention exhibit at least about 14% increased sensitivity compared to conventional digital PCR using paired PCR primers for amplification.

Fig. 1 is a diagram showing the process of the method of the present invention.

Nucleic acid sample

A DNA sample (e.g., cfDNA sample) can be obtained from any sample containing a particular target nucleic acid to be detected, e.g., a bodily fluid or tissue, e.g., blood, urine, skin, hair, stool, and mucus, or an environmental sample, e.g., a water sample or a food sample. The sample may be processed by conventional procedures (e.g., phenol-chloroform extraction or Qiagen kits) to isolate and purify DNA therefrom. The methods of the invention are useful for DNA and RNA targets. For DNA samples, DNA polymerases can be used directly for amplification. For RNA samples, a reverse transcription step using reverse transcriptase is first required.

End repair and A-tail processing

The DNA fragments in the sample are end-repaired and an "A" is added to each 3 'end to provide 3' A overhanging DNA fragments. The end repair and A-tail processing steps can be performed using conventional methods or kits, e.g.Ultra End Repair/dA-lifting Module (NEB, E7442S/L).

Homogeneous adaptors for T/U oligo-primer PCR (TOP-PCR) amplification

Homogeneous adaptors were designed for TOP-PCR amplification. In the homogeneous adaptor, one strand is called a T/U oligonucleotide, with an additional thymine or uracil nucleotide (T/U) at the 3' end; the other strand, called the P oligonucleotide, has a phosphate group at the 5 'end, the 3' end nucleotide of which has no excess T or U. The adapter may be blunt-sticked (i.e., one end is blunt and the other end is sticked) or double-sticked (i.e., both ends are sticked) adapters. In some embodiments, for "blunt-ended" adaptors, the P oligonucleotide is one base shorter and complementary to the T/U oligonucleotide except at the 3' end of the T/U oligonucleotide at the T/U. In some embodiments, the P oligonucleotide is longer than the T/U oligonucleotide for "double-sticky" adaptors. Homogeneous adaptors as used herein require (i) that the T/U oligonucleotide have additional 3' -T/U (i.e., a "T" or "U" overhang at the 3' end) and no 5' -phosphate; (ii) the P oligonucleotide requires a 5' -phosphate; and (iii) the T/U oligonucleotide is complementary to the P oligonucleotide except for the 3' -T/U overhang of the T/U oligonucleotide. The length and sequence of the T/U oligonucleotide and the P oligonucleotide may vary. In some embodiments, the P oligonucleotide sequence is 5'-GTCGGAGTCTgcgc-3' (SEQ ID NO:24) and the T/U oligonucleotide sequence is 5 '-AGACTCCGAC(T) -3' (SEQ ID NO: 23). In some embodiments, the P oligonucleotide sequence is 5'-GT CGG AGT CTA GCG CT-3' (SEQ ID NO:2) and the T/U oligonucleotide sequence is 5'-AGC GCT AGA CTC CGA CT-3' (SEQ ID NO: 1). Furthermore, in some embodiments, "3 '-U" may be used instead of "3' -T", and the double-stranded half-adaptor (HA) may be completely trimmed away after amplification by using "user enzyme" (Uracil-Specific Excision Reagent, NEB).

Ligation of homogeneous adaptors to DNA fragments

After the end repair and A-tail processing steps, homogeneous adaptors are ligated to both ends of the 3 'A-overhang DNA fragments to produce adaptor-ligated DNA fragments in which the 3' -T/U of the T/U oligonucleotide of the adaptor is complementary to the 3'-A overhang of the 3' -A overhang DNA fragments. The ligation can be performed under appropriate conditions, e.g., overnight at about 25 ℃ in a thermocycler in an appropriate ligation mixture containing adaptors, 3' -A protruding DNA fragments, ligase, and ligation buffer. The ligation mixture can be directly subjected to PCR amplification with or without DNA purification.

PCR/detection reagent

Connection ofThereafter, the sample is combined with PCR/detection reagents to provide an amplifiable/detectable sample. PCR reagents typically include primers, nucleotides, polymerase, and buffers. The detection reagent typically includes one or more detectable probes. These input reagents may be provided as separate reagents added separately to the sample, or some or all of the reagents may be provided as a mixture of reagents added in a pre-mixed form to the sample. The PCR reagents typically include buffers selected to facilitate the amplification reaction. Magnesium ions, e.g. MgCl₂Usefully contained in a buffer. The PCR reagents also include nucleotides. Four dNTPs (dATP, dCTP, dGTP, and dTTP) are typically provided at equimolar concentrations. Various PCR polymerases can be used in the same amplification. Suitable polymerases will generally have optimal activity at about 75 ℃ and the ability to retain that activity after prolonged action (e.g., at temperatures above 95 ℃). Useful polymerases can include, e.g., Taq DNA polymerase, e.g. Stoffel fragment of (c), etc. In particular, the PCR reagents include a single primer having the nucleic acid sequence of the T/U oligonucleotide strand as described herein as forward and reverse primers for amplification. A detection reagent comprising a probe specific for the target nucleic acid may be added to the sample and a detectable signal, such as a fluorescent signal caused by degradation of the probe, may be detected.

Partition/dilution (dilution)

The sample ready for amplification/detection is divided into multiple partitions, each containing a limited number of copies of adaptor-ligated NA fragments. In particular, most partitions may not contain any replications, other partitions may contain only one replication, other partitions may contain two replications, three replications, or even a greater number of replications. The replication of each partition may be adjusted as needed. In some embodiments, the partitioning is performed to the extent that more than 50% of the partitions contain no more than one duplicated adaptor-ligated NA fragment. In some embodiments, the partitions are made to the extent that each partition contains at least one replicated adaptor-ligated NA fragment, e.g., 1-5 replicates per partition. Partitioning can be performed in emulsion droplets or in a porous medium as known in the art, for example, as described in Lodrini et al, 2017, and U.S. patent application publication nos. 2009/0053719 and 20150099644, which are incorporated herein by reference. In some embodiments, the sample is divided into multiple small reactions in oil droplets by an oil-in-water emulsion technique. Oil droplets are generated using a droplet generator. Typically, approximately 20,000 oil droplets are formed from each 20 microliter sample.

T/U oligonucleotide-based PCR amplification

After partitioning, PCR amplification was performed using free T/U oligonucleotides as the only PCR primers. As used herein, a free T/U oligonucleotide is a single primer having the nucleic acid sequence of a T/U oligonucleotide strand as described herein. A free T/U oligonucleotide refers to a T/U oligonucleotide that is not formed in the adaptor with its complementary P oligonucleotide. In this way, all DNA fragments ligated to adapters at both ends are amplified in equal proportion.

Detection/quantification

Detection of the target DNA can be performed by a number of methods known in the art, such as flow cytometry using fluorescent probes.

In certain embodiments, a probe having a detectable label, such as a fluorophore (e.g., FAM, 6-fluorescein imide), is used for detection. The fluorescent probe has a complementary sequence that specifically hybridizes to the target nucleic acid fragment, wherein the fluorophore is released from the probe when the target nucleic acid fragment is amplified by PCR (generating a positive signal indicating a detected sequence), and wherein the fluorophore is not released from the probe if the target nucleic acid fragment is not present or amplified (generating a negative signal indicating no detected sequence). In some embodiments, a detectable probe is present in the PCR mixture.

Conventional digital PCR uses probes that are typically designed based on a specific site (e.g., mutation location) in the internal region of the target nucleic acid that is amplified using paired primers; such probes cannot cover all genetic variations, thus limiting the detection of other potential biomarkers in nucleic acids. In contrast, the method of the present invention allows for amplification of all nucleic acid fragments in a sample, and thus shotgun probes capable of specifically hybridizing to any region within the target nucleic acid can be used, and detection of the target nucleic acid can be achieved by increased sensitivity. Please refer to a specific embodiment of fig. 2. According to the present invention, not only fragment "g" (covering both primer binding sites), but also other fragments "a" to "f" (having only one primer binding site or even no primer binding site), can detect nucleic acids all originating from the same target pathogen, for example using shotgun probes with detectable labels capable of specifically hybridizing to any region within the target DNA, thereby increasing detection sensitivity and minimizing false negatives.

After detection, the number of positive partitions (detected sequences) relative to negative partitions (undetected sequences) is counted to determine an estimated amount of target nucleic acid fragments in the sample. Quantification can be performed according to methods known in the art, e.g., as described by Lodrini et al, 2017. In certain embodiments, the PCR amplified droplets can be measured in a QX200ddPCR droplet reader and analyzed for target copy number using QuantaSoft analysis software.

Also provided are kits for performing the methods of detecting nucleic acid fragments in a sample as described herein. Specifically, the kit comprises

(i) An adaptor ligation reagent comprising a homogeneous adaptor, a ligation buffer, and a ligase, wherein the homogeneous adaptor comprises a P oligonucleotide strand with a 5 '-phosphate and a T/U oligonucleotide strand with a 3' -T or 3-U without a 5 '-phosphate, wherein the T/U oligonucleotide strand is complementary to the P oligonucleotide strand except at the 3' -T or 3'-U of the T/U oligonucleotide strand, wherein the homogeneous adaptor is capable of ligating to the nucleic acid fragments at both ends, wherein the nucleic acid fragments have 3' -A overhangs;

(ii) PCR reagents including a single primer having the nucleic acid sequence of the T/U oligonucleotide chain, dNTPs (dATP, dCTP, dGTP, and dTTP), PCR buffer, and DNA polymerase; and

(iii) a detection reagent comprising one or more detectable probes having a complementary sequence that specifically hybridizes to the nucleic acid fragment.

In some embodiments, the kit further comprises instructions for use. In particular, the instructions for use comprise instructions for carrying out the method of the invention, comprising steps (a) to (g).

Practicality and advantages of the invention

The methods of the invention can be used for diagnosis or prognosis, particularly in cfDNA-based assays. Non-invasive methods of detecting body fluid samples containing cfDNA have been described which can be used for the diagnosis of gene defects, infectious agents and diseases, particularly for early detection and at least prognosis, as cfDNA is available even after diseased tissue, such as tumors, has been removed. However, conventional PCR, including qPCR or dPCR, is designed to be template dependent, requires at least one pair of primers, and is therefore not suitable for cfDNA detection, since cfDNA as a template is generally not high in quality and quantity, and therefore limited in sensitivity, and if the number of PCR cycles increases, deviations may occur. The method of the present invention, by using a homogeneous adaptor composed of a P oligonucleotide and a T/U oligonucleotide, ligated to DNA, and the T/U oligonucleotide as a single primer, can amplify all DNAs in a sample in an equal proportion to any initial amount, and can detect target DNA using a specific probe to increase sensitivity without generating a significant deviation (false negative).

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples

Although digital PCR (dpcr) is a powerful method for analyzing genetic mutations and Copy Number Variations (CNVs), it is not suitable for cell-free dna in clinical samples (cfdnas) analysis because of the small number of cfDNA fragments and the high fragmentation, which may cause the conventional digital PCR method using double primer amplification to generate false negatives. To solve this problem, we developed droplet digital T/U oligo-polymerase chain reaction (ddTOP-PCR), which relies on adaptors to amplify all cfDNA fragments without differentiation, followed by detection using an oligomerizing probe labeled FAM or HEX, which generates chromophores from specific targets during extension. The fluorescent signal was then detected by a QX200ddPCR instrument. The results show that ddTOP-PCR is able to detect N-myc sequences with a 5' primer binding site deletion, whereas ddPCR is not. Further testing of samples containing 5' primer binding site deleted constructs and/or fragments of dual primer binding site intact N-myc constructs revealed that the sensitivity of ddTOP-PCR was improved by about 14% compared to conventional ddPCR, despite the reduced signal intensity. These proof-of-concept verifications demonstrate that ddTOP-PCR is superior to its relative approach in analyzing cfDNA in liquid biopsies.

1. Materials and methods

1.1. Cloning of N-myc sequences as analytical standards

To optimize experimental conditions and provide criteria for analysis of copy number variation, vectors carrying the N-myc gene sequence were constructed based on the primary combination of human chromosome 2 GRCh38.p12 (accession No.: NC-000002.12) retrieved from the NCBI database using primers directed against the human N-myc sequence. An N-myc amplicon (157bp) was cloned/amplified from the genomic DNA of the Be2C cell strain using forward primer 5'-AAG GGG TGC TCT CCA ATT CT-3' (SEQ ID NO:13) and reverse primer 5'-CGG TTT AGC CAC CAA CTT TC-3' (SEQ ID NO: 14). NAGK amplicon (172bp) was cloned/amplified from the genomic DNA of Be2C cell line using forward primer 5'-CCC CTT TCC CGC TAT ATC TT-3' (SEQ ID NO:15) and reverse primer 5'-ATG CAG GGT TTG ATG GGA TA-3' (SEQ ID NO: 16). Polymerase Chain Reaction (PCR) was performed using Q5 High-Fidelity 2X Master Mix (NEB, Ma., USA) to amplify the target amplicon. PCR reactions were performed in a mixture (50. mu.l) containing the corresponding primer pair, Be2C genomic DNA (10ng), 1x Q5 High-Fidelity Master Mix. The mixture was then exposed to 98 ℃ for 1 minute, then to 98 ℃ for 20 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 10 seconds for 30 cycles. The mixture was then allowed to act at 72 ℃ for 2 minutes for final extension. The PCR products were then analyzed and amplicons of the expected size were extracted from 2% agarose gels using the QIAquick gel extraction kit (Qiagen, NW, germany) according to the instruction manual, followed by gel DNA extraction.

1.2 ligation of half-adaptors (HA) to N-myc and NAGK standard templates

Half-adaptors (HA) were first prepared by annealing 16 monomer units of P oligonucleotide (5 '-pGTCGGAGTCTAGCT-3C 6-3') (SEQ ID NO:2) and 17 monomer units of T/U oligonucleotide (5 '-AmC 6-AGCGCTAGACTCCGACT-3') (SEQ ID NO:1) at a molar ratio of 1:1 at 95 ℃ for 5 minutes, and then gradually reducing the temperature to 4 ℃ using a thermal cycler.

Before ligation, for Illumina sequencingUltra^TMII DNA library preparation kit (NEB, Ma., USA) was modified slightly by first performing end repair and 3' A tail processing on 10ng of 157bp N-myc amplicon and 172bp NAGK amplicon. Then, ligation is performed using, for example, a 50:1 ratio of HA to amplicon, and the reaction is allowed to proceed overnight in a thermocycler at 16 ℃.

If desired, the ligation mixture was subjected to direct TOP-PCR without purification. The reaction was performed in a mixture (50. mu.l) using 1. mu.M T/U oligonucleotide (5'-AGCGCTAGACTCCGACT-3') (SEQ ID NO:1), ligation mixture (5. mu.) and 1 XPhusion HF reaction buffer (Thermo Fisher Scientific, Ma., USA) containing Mg²⁺(1.5mM), Phusion high fidelity hot start DNA polymerase (1U), and dNTPs (1 mM). The mixture is then allowed to act at 98 ℃ for 1 minute, then at 98 ℃ for 20 seconds, at 57 ℃ for 30 seconds, and at 72 ℃ for 1 minute for sheared gDNA, against a standard modelThe plate was exposed for 10 seconds for 30 cycles. The mixture was then allowed to act at 72 ℃ for 5 minutes for final extension. N-myc and NAGK amplified by TOP-PCR, i.e., HA-N-myc-HA and HA-NAGK-HA, respectively, were purified using the QIAquick PCR purification kit (Qiagen, NW, Germany) according to the instructions and quantified using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Mass., USA).

1.3 construction of Standard formwork

To test the T/U oligonucleotides for probe specificity. Different ratio combinations of pHE-N-myc-HA and pHE-NAGK-HA templates were prepared. The ratio of 10-fold sequence dilution of pHE-N-myc-HA to pHE-NAGK-HA template was prepared at 100:1 to 1: 100. The ddPCR mixture was prepared in a slightly modified manner. Prior to droplet generation, the ddPCR reaction (20. mu.l) included T/U oligonucleotide primer (8. mu.M), Mpb1+2 (0.25. mu.M), Npb2 (0.25. mu.M), DNA template (4.0. mu.l), 1 XDdPCR^TMProbe ultra mix (no dUTP) (Bio-Rad, ca, usa). A mixture of ddPCR droplets was prepared and the ddPCR reaction was performed as described above.

1.4 testing of gDNA-HA

Specific amplification of N-myc from the NAGK target in gDNA mixtures was performed using ddTOP-PCR. The constructed gDNA-HA template was used in this experiment using the above ddPCR parameters. The experiment used a 10-fold sequence dilution of sheared gDNA-HA at varying input volumes ranging from 100ng to 100 pg. Prior to droplet generation, the ddPCR reaction (20. mu.l) contained T/U oligonucleotide primers (8. mu.M), Mpb1+2 (0.25. mu.M), Npb2 (0.25. mu.M), gDNA-HA template (100ng to 100pg), 1 XDddPCR^TMProbe ultra mix (no dUTP) (Bio-Rad, ca, usa). A mixture of ddPCR droplets was prepared and the ddPCR reaction was performed as described above.

1.5 construction of vectors with HA-N myc-HA and HA-NAGK-HA sequences as analytical standards

HA-N myc-HA and HA-NAGK-HA constructs were then cloned using the HE Swift cloning kit (Toolbiotech, Taiwan, China), transformed into DH5 α competent cells, and plated on ampicillin LB agar plates. Bacterial colonies were screened and sequenced by Sanger sequencing to verify the sequence after plastid extraction using the QIAprep Spin Miniprep kit (Qiagen, NW, germany).

The standard template for ddTOP-PCR was generated by amplifying recombinant plasmids with the correct HA-Nmyc-HA and HA-NAGK-HA sequence in a 100. mu.l PCR reaction containing the pHE-F primer (5'-CGA CTC ACT ATA GGG AGA GCG GC-3'; SEQ ID NO:17, 0.5. mu.M), pHE-R primer (5'-AA GAA CAT CGA TTT TCC ATG GCA G-3'; SEQ ID NO:18, 0.5. mu.M), DNA (1ng), 1x Q5 High-Fidelity Master Mix. The mixture was then allowed to act at 98 ℃ for 1 minute, then at 98 ℃ for 20 seconds, 64 ℃ for 30 seconds, and 72 ℃ for 10 seconds for 30 cycles. The mixture was then allowed to act at 72 ℃ for 2 minutes for final extension. pHE-HA-N myc-HA and pHE-HA-NAGK-HA PCR amplicons of 309bp and 325bp sizes, respectively, were purified and quantified using a QIAquick PCR purification kit (Qiagen, NW, Germany).

1.6 construction of 5' deleted N-myc and NAGK constructs as analytical standards

The N-myc 5' primer binding site deleted construct was amplified from Q5 High-Fidelity Master Mix with forward primer (5'-AGC GCT AGA CTC CGA CTT CAC TAA AGT TCC TTC CAC CCT CTC CTG GGG AG-3') (SEQ ID NO:19) and reverse primer (5'-AGC GCT AGA CTC CGA CTT AGC CAC CAA CTT TCT CCA ATT TTA TTC CTC AG-3') (SEQ ID NO: 20). The construct with the deletion of the 5' primer binding site of NAGK was amplified from Q5 High-Fidelity Master Mix with forward primer (5'-AGC GCT AGA CTC CGA CTG TGT TGC CCG AGA TTG ACC CGG TGA GTT GAG GT-3') (SEQ ID NO:21) and reverse primer (5'-AGC GCT AGA CTC CGA CTA TGC AGG GTT TGA TGG GAT AGT CCC ATC-3') (SEQ ID NO: 22). HA-N myc-F del-HA and HA-NAGK-F del-HA PCR amplicons of 160bp and 146bp, respectively, were purified and quantified using QIAquick PCR purification kit (Qiagen, NW, Germany).

1.7 PCR primers for amplification

The paired primer sequences for ddPCR were obtained from Lodrini et al, but the sequences were slightly modified (Lodrini et al, 2017). Primers and probes were synthesized by Integrated DNA Technology (IDT). In fact, for amplification of the N-myc sequence, a forward primer (5'-GTG CTC TCC AAT TCT CGC CT-3') (SEQ ID NO:3) and a reverse primer (5'-GAT GGC CTA GAG GAG GGC T-3') (SEQ ID NO:4) were used.

1.8 Probe for detection

To detect N-myc amplification, 3 probes were designed (as shown below). These include 1) probe Mpb1(FAM-N-myc probe)/56-FAM/CAC TAA AGT/ZEN/TCC TTC CAC CCT CTC CT/3IABKFQ/(SEQ ID NO: 10); 2) probe Mpb1+1(FAM-N-myc probe +1 nt): 56-FAM/CAC TAA AGT/ZEN/TCC TTC CAC CCT CTC CTG/3IABKFQ/(SEQ ID NO: 11); 3) probe Mpb1+2(FAM-N-myc probe +2 nt): 56-FAM/CAC TAA AGT/ZEN/TCC TTC CAC CCT CTC CTG G/3IABKFQ/(SEQ ID NO: 12). The initial test used probe Mpb1+ 1.

1.9 conditions of ddPCR and ddTOP-PCR

In order to achieve the feasible state of ddTOP-PCR and ultimately optimize its sensitivity and specificity, the length of PCR primers and fluorescent probes must be tested as well as the experimental conditions. In addition, it is necessary to run a conventional ddPCR simultaneously as a positive control for the initial setup, so both optimization and experimental conditions apply to ddTOP-PCR and ddPCR.

From the preliminary results, we determined that a primer concentration of 0.9. mu.M was used for the gene-specific primer (ddPCR control) and a primer concentration of 32. mu.M was used for the T/U oligonucleotide (ddTOP-PCR). A total of 20. mu.l of PCR reaction contained one or more primers (either the paired primers for ddPCR control or the T/U oligonucleotide primers for ddTOP-PCR), 0.25. mu.M probe, DNA template (2.0. mu.l), 1 XDdPCR probe superscocktail (without dUTP) (Bio-Rad). The droplets were generated by mixing the prepared PCR reaction mixture (20. mu.l) with 70. mu.l of a droplet digital PCR oil (Bio-Rad Co.). A total of 40. mu.l of ddPCR droplet mix was transferred to a 96-well plate and sealed before PCR reaction in a Bio-Rad T-100 thermocycler (Bio-Rad Co.). Both ddPCR and ddTOP-PCR preparations were then placed in a Bio-Rad PCR instrument for amplification and chromophore generation under the following conditions: 10 minutes at 95 ℃ followed by 40 cycles of 30 seconds at 94 ℃ and 60 seconds at 58 ℃. The reaction was stopped by exposure to 98 ℃ for 10 minutes. After the PCR reaction, the positive droplets were quantified using a QX200ddPCR droplet reader and analyzed using QuantaSoft analysis software (version 1.7.4, Bio-Rad).

2. Results

2.1 detection of N-myc Gene sequences for comparison Using ddPCR and ddTOP-PCR

Initial tests were conducted to demonstrate that the concept of improved sensitivity, expressed as an increase in positive counts in the QX200ddPCR instrument, should be achieved by replacing conventional PCR with TOP-PCR.

Samples containing only NAGK or N-myc sequences provide an easily verifiable system to improve conditions under which experimental conditions (e.g., sample preparation, PCR component concentrations, and reaction conditions) can be adjusted based on experimental results.

For testing, we prepared two types of fragments containing N-myc sequences: one with two primer binding sites and the other with a 5' primer binding site deleted, only the 3' binding site was retained, and a control with NAGK lacking the 5' primer binding site (table 1).

TABLE 1 Experimental design

We used a QX200ddPCR instrument for a different procedure, ddPCR or ddTOP-PCR, to demonstrate that ddTOP-PCR has the potential to detect fragments with defective primer binding sites, which may be present in cfNA sample pools.

For proof of concept testing, we prepared samples containing variable percentages of the above-described 5' primer binding site deletion templates. The initial total input per sample was calculated to be approximately 12,000 replicates and all experiments were performed by a QX200ddPCR instrument using the settings reported by Lodrini et al, with minor modifications (Lodrini et al, 2017). The same N-myc oligonucleotide probe sequence used by Lodrini et al was also used in this experiment.

The results show that, in general, the ddTOP-PCR method using T oligonucleotide as the only primer for amplification counts higher than the ddPCR method using dual internal primers (FIG. 3).

To compare the sensitivity of ddTOP-PCR with ddPCR, we estimated the copy number input (using Qubit) in the original sample and the corresponding detected copy number, and then calculated the percentage detected (Table 2).

Table 2 comparison of sensitivity between ddPCR and ddTOP-PCR.

The results showed that ddPCR was able to detect about 48.6% of template, while ddTOP-PCR was able to detect about 58.5% -62.8% of template, which indicates a 10% -15% improvement in sensitivity from ddPCR to ddTOP-PCR. Note that while accuracy is affected by variations/variations due to factors such as the quantization equipment (e.g., Qubit), personal technology, the QX200 machine itself, etc., the general trend for each approach has been to some degree of reliability.

Calculating the standard deviation

However, the higher sensitivity of ddTOP-PCR was affected by more scattered signal intensity (FIG. 4). Most of the positive signal intensity in ddTOP-PCR droplets was lower than in ddPCR droplets. Presumably due to the shorter and well-defined amplification window resulting from the use of dual internal primers, which in turn resulted in a higher uniformity of signal for ddPCR than for ddTOP-PCR, where amplification started with flanking adapters, which are far from the double primer position.

As shown for all mixed samples of FIG. 4, the color intensity of droplets produced from ddTOP-PCR can be higher or lower than the color intensity of droplets produced from ddPCR and more dispersed. As shown in lane 4, ddPCR did not detect the 5' primer binding site deleted fragment, which was efficiently detected by ddTOP-PCR. The first two lanes in the figure indicate that the false positives for NAGK template are low. Previous observations showed that the blank background had a clean count (0) for both ddTOP-PCR and ddPCR methods (data not shown).

Amplification of the internal double primer, as shown by ddPCR, produced more uniform results, while on the other hand amplification of adapter-based ddTOP-PCR had higher sensitivity but lower intensity.

These data also indicate the need for further optimization. Also, shorter fragments appear to have advantages over longer fragments.

3. Conclusion

This proof-of-concept study provided preliminary data to demonstrate the development of ddTOP-PCR and experimentally demonstrate the feasibility of using ddTOP-PCR to improve the accuracy of cfDNA-based Copy Number Variation (CNV), gene mutations, and disease gene expression and analysis of SNP changes.

Compared to conventional ddPCR, ddTOP-PCR has many advantages: 1) digital PCR is not suitable for cfDNA analysis because, as a template-dependent method, traditional PCR requires two primer binding sites to be present together in the same fragment. On the other hand, ddTOP-PCR, which is an adaptor-dependent PCR method, does not have such a limitation and thus can detect a partial fragment. 2) Before ddTOP-PCR experiments, TOP-PCR alone could be used to preserve low quantitative samples, whereas traditional PCR did not. Since the number of cfDNA samples can be very small, there must be a good method to recruit all cfDNA fragments in the analysis. Therefore, it is very important to store minute DNA fragments without distinction. 3) ddTOP-PCR may also be suitable for early detection of cancer and other diseases, whereas traditional ddPCR does not.

Cell-free DNA fragments were heavily diluted with non-specific DNA fragments derived from normal or non-diseased cells. Conventional designs with a single DNA fragment per droplet are not cost effective and therefore impractical. Conversely, multiple copies per droplet are more suitable for cfDNA. We started with approximately 4 replicated DNA fragments per droplet to perform initial testing of ddTOP-PCR in a QX200 instrument.

The present results also show that further improvements in dosing devices and methods would be helpful.

Reference to the literature

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Dong,L.,Meng,Y.,Sui,Z.,Wang,J.,Wu,L.,and Fu,B.(2015).Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.Sci Rep 5,13174.

Hindson,B.J.,Ness,K.D.,Masquelier,D.A.,Belgrader,P.,Heredia,N.J.,Makarewicz,A.J.,Bright,I.J.,Lucero,M.Y.,Hiddessen,A.L.,Legler,T.C.,et al.(2011).High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.Anal Chem 83,8604-8610.

Lodrini,M.,Sprussel,A.,Astrahantseff,K.,Tiburtius,D.,Konschak,R.,Lode,H.N.,Fischer,M.,Keilholz,U.,Eggert,A.,and Deubzer,H.E.(2017).Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma.Oncotarget 8,85234-85251.

Morley,A.A.(2014).Digital PCR:A brief history.Biomol Detect Quantif 1,1-2.

Nai,Y.S.,Chen,T.H.,Huang,Y.F.,Midha,M.K.,Shiau,H.C.,Shen,C.Y.,Chen,C.J.,Yu,A.L.,and Chiu,K.P.(2017).T Oligo-Primed Polymerase Chain Reaction(TOP-PCR):A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids.Sci Rep 7,40767.

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Sequence listing

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<213> Artificial sequence

<220>

<223> HA-NAGK-F del-HA

<400> 9

agcgctagac tccgactgtg ttgcccgaga ttgacccggt gagttgaggt gggagtgaag 60

gtggggagct gctgggtgag gagtggtcct ttcccactgt ggatgggact atcccatcaa 120

accctgcata gtcggagtct agcgct 146

<210> 10

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Probe Mpb1

<400> 10

cactaaagtt ccttccaccc tctcct 26

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Probe Mpb1+1

<400> 11

cactaaagtt ccttccaccc tctcctg 27

<210> 12

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Probe Mpb1+2

<400> 12

cactaaagtt ccttccaccc tctcctgg 28

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> N myc Forward primer (amplicon primer)

<400> 13

aaggggtgct ctccaattct 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> N myc reverse primer (amplicon primer)

<400> 14

cggtttagcc accaactttc 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> NAGK Forward primer (amplicon primer)

<400> 15

cccctttccc gctatatctt 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> NAGK reverse primer (Complex primer)

<400> 16

atgcagggtt tgatgggata 20

<210> 17

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> pHE-F primer

<400> 17

cgactcacta tagggagagc ggc 23

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> pHE-R primer

<400> 18

aagaacatcg attttccatg gcag 24

<210> 19

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> N myc Forward primer (5' del)

<400> 19

agcgctagac tccgacttca ctaaagttcc ttccaccctc tcctggggag 50

<210> 20

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> N myc reverse primer (5' del)

<400> 20

agcgctagac tccgacttag ccaccaactt tctccaattt tattcctcag 50

<210> 21

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> NAGK Forward primer (5' del)

<400> 21

agcgctagac tccgactgtg ttgcccgaga ttgacccggt gagttgaggt 50

<210> 22

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> NAGK reverse primer (5' del)

<400> 22

agcgctagac tccgactatg cagggtttga tgggatagtc ccatc 45

<210> 23

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> T oligonucleotide (11 monomer units)

<400> 23

agactccgac t 11

<210> 24

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> P oligonucleotide (14 monomer units)

<400> 24

gtcggagtct gcgc 14