Method for obtaining fragmented DNA single-strand pool and application thereof

文档序号：900180 发布日期：2021-02-26 浏览：10次中文

阅读说明：本技术 一种获得片段化dna单链池的方法及其应用 (Method for obtaining fragmented DNA single-strand pool and application thereof ) 是由齐浩郜艳敏于 2020-11-11 设计创作，主要内容包括：本发明涉及生物技术领域,本发明公开了一种获得片段化DNA单链池的方法及其应用。本发明利用只有一条靠近待检测位点的引物且在ddNTPs存在的情况进行PCR扩增片段化DNA,产生长度较短的末端带有一个ddNTP的DNA单链池,使得扩增的片段长度集中在80-120nt,对比不加ddNTPs的方法,本发明使得片段化DNA更充分更全面地扩增出带有待检测位点的短的单链DNA片段,产生的片段长度更短,长度分布更窄。同时在后续的应用Blocker介导的锁式探针技术以及其他杂交探针进行片段化DNA的检测时,检测更准确和更灵敏,能够应用于片段化DNA的相关检测中。(The invention relates to the technical field of biology, and discloses a method for obtaining a fragmented DNA single-strand pool and application thereof. The invention utilizes only one primer close to the site to be detected and carries out PCR amplification fragmentation DNA under the condition that ddNTPs exist, a DNA single-chain pool with a short length and a ddNTP at the tail end is generated, so that the length of the amplified fragments is concentrated in 80-120nt, compared with a method without ddNTPs, the invention ensures that the fragmentation DNA more fully and comprehensively amplifies short single-chain DNA fragments with the site to be detected, the length of the generated fragments is shorter, and the length distribution is narrower. Meanwhile, when the Blocker-mediated padlock probe technology and other hybridization probes are subsequently applied to the detection of fragmented DNA, the detection is more accurate and sensitive, and the method can be applied to the related detection of fragmented DNA.)

1. A method of obtaining a pool of single strands of fragmented DNA, comprising:

step 1, 5' phosphorylation of fragmented DNA; designing a single complementary primer aiming at a target gene site to be detected, wherein the 5 'end of the single complementary primer and a target gene binding site are positioned on one side of the 3' end of the site to be detected by 10-30 bp;

step 3, taking the 5' phosphorylated fragmented DNA in the step 1 as a template, and performing PCR amplification by using the single complementary primer; wherein ddNTPs are required to be added into a PCR amplification system;

and 4, adding exonuclease to degrade the template after PCR amplification is finished, and obtaining a fragmented DNA single-strand pool.

2. The method of claim 1, wherein the single complementary primer is present in the PCR amplification system at a concentration of at least 0.2 μ M.

3. The method of claim 1, wherein the ratio of ddNTPs to dNTPs in the PCR amplification system is (0.02-50): 1.

4. Use of the method of any one of claims 1 to 3 for detecting single-site variations in fragmented DNA.

5. A method for detecting single site variation in fragmented DNA, comprising:

step 1, obtaining a single-stranded pool of fragmented DNA by the method of any one of claims 1 to 3;

and 2, analyzing the fragmented DNA single-strand pool by adopting the conventional detection method.

6. The method of claim 5, wherein the existing detection method is a padlock probe technique combined with an RCA amplification technique.

7. The method of claim 6, wherein the padlock probe technique is combined with RCA amplification technique as follows:

step 1, designing a padlock probe and a wild type blocker probe aiming at a wild type single-chain target gene; designing a mutant type blocker probe aiming at the mutant type single-chain target gene;

step 2, carrying out RCA amplification on the fragmented DNA single-chain pool by using the three probes in the step 1 to obtain a Ct value which is A3;

performing RCA amplification on the fragmented DNA single-chain pool by using the lock type probe and the wild type blocker probe in the step 1 to obtain a Ct value which is A2;

carrying out RCA amplification on the fragmented DNA single-chain pool by using the lock probe in the step 1 to obtain a Ct value which is A1;

wherein the wild type/mutant type blocker probe is a single-stranded DNA oligonucleotide, is divided into a region sequence a and a region sequence b in the 3 '→ 5' direction, is divided into a region sequence I-III, and can be connected with a DNA polymerase in the sequence I → II → III → I to form a ring 3 'and 5'; wherein, the I, III region sequence is complementary with the target gene, the b region sequence is the same as the I region sequence, and the a region sequence is complementary with the wild type/mutant target gene; the gibbs free energy of binding of the a region sequence to the target gene is equal to the gibbs free energy of binding of the III region sequence to the target gene; the position of single-site variation of the fragmented DNA is positioned on a target gene sequence which is complementary to the sequence of the a region;

step 3, calculating D value according to formula D ═ A3-a2)/(A3-a1), and D value represents the ratio of single site variation of fragmented DNA.

8. The method of claim 7, wherein the length of the a-region sequence is 2 to 20 nt.

9. The method of claim 7, wherein the b region sequence length and the I region sequence length are independently selected from 4 to 30 nt.

10. The method of claim 7, wherein the length of the region III sequence is 2 to 20 nt.

11. The method of claim 7, wherein the length of the region II sequence is 15 to 45 nt.

Technical Field

The invention relates to the technical field of biology, in particular to a method for obtaining a fragmented DNA single-strand pool and application thereof.

Background

Cancer is a common malignant tumor in clinic, the disease has high rate of rapid progress and high mortality, and causes great harm to the psychology and the economy of patients, and the cancer is also a public health problem which is concerned by people. In recent decades, cancer diagnosis is a topic of research, and in 2013, a paper that proves that most of cancers suffered by human are caused by 21 main gene mutations is published in Nature, so far, gene detection becomes an important means for cancer treatment and prevention. Through gene detection, the genes of the variation of the human body can be found out, and chemotherapeutic drugs or molecular targeted therapeutic drugs are selected according to the gene detection result, so that the personalized treatment of the cancer is realized.

In gene detection, cfDNA detection, monitoring and molecular research provide a meaningful and noninvasive method for early disease detection. Currently, fragmented DNA (cfDNA) Circulating in human body fluids is of interest to numerous scientists and clinicians as genetic material for non-invasive screening and diagnosis. Fernando M R and coworkers found that the plasma contained 76bp,135bp,490bp and 905bp of DNA at 100%, 39%, 18% and 5.6%, respectively, indicating that these free DNAs were highly fragmented. However, highly fragmented DNA is very challenging for currently available nucleic acid detection techniques.

Most of the current detection methods detect gene mutation of single-stranded dna (ssdna), while cfDNA in peripheral blood is fragmented double-stranded dna (dsdna), so it is more critical to amplify a single-stranded form of cfDNA fragment with a detection site as completely as possible.

Currently, the method for obtaining ssDNA can be performed by a Lambda exonuclease degradation method, i.e., firstly, a phosphorylated primer and a common primer are used for typical PCR amplification to obtain a double-stranded product, and then, Lambda exonuclease (which recognizes phosphate groups and degrades phosphate-group-containing strands into single nucleotides one by one) is added for degradation, so as to obtain single-stranded DNA. However, this method loses part of the sample when acquiring single strands of cfDNA because cfDNA is highly fragmented and is currently poorly understood by scientists, knowing only the distribution range of its length, but the location of the genome at which it is fragmented is uncertain. Traditional PCR cannot design a set of primers to obtain cfDNA fragment single strands of all kinds. Therefore, if a certain cfDNA fragment cannot simultaneously contain the binding sequences of two primers, the PCR process cannot be completed, and thus a single-stranded form of the cfDNA cannot be obtained, and genetic information carried by the cfDNA is lost in subsequent detection.

And when cfDNA is used as a template, a long single strand may be amplified by the conventional method for obtaining a single strand pool, such as asymmetric PCR, and multiple cfDNA fragments are combined (the long strand is amplified because the product produced in each round can be used as a primer in each subsequent round, and is combined with multiple fragments in the fragmented cfDNA to generate a long strand). The longer ssDNA is easy to form a secondary structure, so that the sensitivity and the accuracy of a later detection method are reduced.

In conclusion, the cfDNA single strand obtained by the common PCR method, which is the Lambda exonuclease degradation method, cannot show all genetic information of multiple types of fragmented cfDNA to-be-detected sites, and byproducts with complex secondary structures are easily generated, thereby finally reducing the sensitivity and accuracy of the subsequent detection method.

Disclosure of Invention

In view of the above, the present invention provides a method for obtaining a single-stranded pool of fragmented DNA, which can exhibit multiple forms of fragmented single-stranded DNA, and avoid loss of a site to be detected of the fragmented single-stranded DNA; meanwhile, the obtained fragmented single-stranded DNA is short in length and not easy to form a secondary structure;

another object of the present invention is to provide the use of the above method for detecting single-site variations in fragmented DNA;

another object of the present invention is to provide a method for detecting single-site mutation in fragmented DNA, which can accurately detect single-site mutation in fragmented DNA using the above method.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method of obtaining a pool of single strands of fragmented DNA, comprising:

and 4, adding exonuclease to degrade the template after PCR amplification is finished, and obtaining a fragmented DNA single-strand pool.

DNA in blood is highly fragmented, particularly cfDNA, leading to diversity and unclear mechanism of DNA fragment species due to different fragmentation sites. Compared with the traditional PCR, the invention is similar to the current asymmetric PCR technology, but only uses one primer, the invention designs the single primer close to the target gene site to be detected to enrich the single-stranded DNA molecules, and can more fully and completely amplify and enrich the short single-stranded DNA fragment with the site to be detected through ddNTPs, thereby avoiding the loss of the fragmented DNA in the amplification process in the traditional PCR and providing a single-stranded DNA template for the subsequent detection of various hybridization probes on the fragmented DNA.

Meanwhile, referring to a schematic diagram of the principle shown in FIG. 1, only a single primer is added, the primer is close to a site to be detected, nucleotides in a PCR system are a mixture of deoxyribonucleotides (dNTPs) and dideoxyribonucleotides (ddNTPs), and when the ddNTPs are added in a polymerization reaction, subsequent polymerization is stopped to generate a DNA single chain to be detected, so that an amplification product is not very long, and the length of the single chain is concentrated to about 100 nt.

Preferably, the concentration of the single complementary primer in the PCR amplification system is at least 0.2. mu.M, and more preferably, the concentration is 0.2-10. mu.M; more preferably, it is at a concentration of 0.2-4. mu.M, 0.2-2. mu.M, or 2-4. mu.M; in a specific embodiment of the invention, the concentration is 0.2. mu.M, 2. mu.M or 4. mu.M;

preferably, the ratio of ddNTPs to dNTPs in the PCR amplification system is (0.02-50): 1. In specific embodiments, the ratio of ddNTPs to dNTPs is 24:1 or 1: 50;

on the premise of the same cfDNA template, the method takes the addition of ddNTPs and the non-addition of ddNTPs as two test groups to detect the influence of the ddNTPs on the method for obtaining the single-chain pool, and the result shows that most of the fragments in the cfDNA single-chain pool generated without the addition of the ddNTPs are concentrated between 100nt and 200nt, most of the fragments in the cfDNA single-chain pool generated by the method are concentrated between 80nt and 120nt, the fragments are obviously shortened, the probability of forming a secondary structure of the generated single chain is greatly avoided, and the detection sensitivity is improved. Moreover, in the method for detecting fragmented DNA single-site variation provided subsequently in the present invention, many mutation samples will be lost in the single-strand pool obtained by the test group without ddNTPs, so that the real mutation rate cannot be presented in the subsequent detection; the mutation rate detected by the invention is obviously higher than that of a test group only added with dNTPs, so that richer mutation conditions can be presented, and the loss is avoided.

Therefore, the invention provides the application of the method for obtaining the single-stranded pool in detecting the single-site variation of the fragmented DNA. Preferably, the fragmented DNA is cfDNA or ctDNA.

Based on the application, the invention also provides a method for detecting the single-site variation of the fragmented DNA, which comprises the following steps:

step 1, obtaining a fragmented DNA single-chain pool by using the method for obtaining the single-chain pool;

and 2, analyzing the fragmented DNA single-strand pool by adopting the conventional detection method.

At present, a plurality of documents report that the padlock probe technology is combined with the RCA amplification technology to detect nucleic acid sequences, miRNA, SNP, single base variation and the like. Preferably, therefore, the existing detection method is a padlock probe technique in combination with an RCA amplification technique.

The detection of nucleic acid sequences, particularly single-site variation, by the padlock probe-RCA technology mainly depends on the base complementary pairing of a probe and a target, and the detection of nucleic acid sequences based on the padlock probe-RCA technology causes certain false positive or false negative due to the problem of ligase fidelity. Taking single base variation detection as an example, the detection of single-site variation by using a padlock probe is basically designed by designing a variation site at the 3 'end of the probe, and the 3' end of the probe is completely complementary and paired with a mutant base and is not matched with the site of a wild type. Ideally, the presence of only the mutant target will form a circular single stranded dna (cssdna). However, at normal temperature, some base pairs are mismatched to some extent, and at this time, the ligase cannot recognize the mismatch, and the 5 '-end phosphate group and the 3' -end hydroxyl group can still be connected to form a phosphodiester bond, so that css DNA is formed (namely, the css DNA is formed in the presence of a wild-type target), and then the RCA reaction is carried out to amplify the signal. This result leads to the occurrence of false negatives, whereas the generation of false positives is exactly the opposite of the generation of false negatives.

Aiming at the problems, the invention provides an improved padlock probe technology combined with an RCA amplification technology, which comprises the following specific steps:

step 2, carrying out RCA amplification on the fragmented DNA single-chain pool by using the three probes in the step 1 to obtain a Ct value which is A3;

performing RCA amplification on the fragmented DNA single-chain pool by using the lock type probe and the wild type blocker probe in the step 1 to obtain a Ct value which is A2;

carrying out RCA amplification on the fragmented DNA single-chain pool by using the lock probe in the step 1 to obtain a Ct value which is A1;

Step 3, calculating a D value according to the formula D ═ A3-a2)/(A3-a1), the D value indicating the ratio of single-site mutations in the fragmented DNA, and a higher D value indicates a higher observed mutation rate.

For both the I and III region sequences of the padlock probe, when they are complementarily bound to the target sequence and not polymerized into a loop by DNA polymerase (i.e., the 3 'and 5' ends are not ligated), the gap between the 3 'and 5' ends is called a breakpoint, which may occur at the I region sequence, at the III region sequence, or just at the split point of the I and III region sequences; the position of the breakpoint is related to the sequence of the blocker probe and is generally located in the middle of the sequence of the I + III region.

For the II region sequence of the padlock probe, different sequences can be designed according to actual requirements; currently, csdna has demonstrated potential clinical applications, such as 2018, j.meng, et.al. designed an artificial circular single-stranded dna (cssd) molecule comprising a succession of sequence sites complementary to miR-9. Thereby leading miR-9 to be hybridized on CSSD through base complementation, further up-regulating the expression of cancer suppressor genes (KLF17, CD17 and LASS2) in vivo and inhibiting the tumor deterioration and lung metastasis. The CSSD molecule is more stable and resistant to degradation than miR inhibitors. Meanwhile, cssDNA was also used for aptamer screening, m.liu.et.al found two high affinity circular DNA aptamers that recognized Glutamate Dehydrogenase (GDH) from Clostridium difficile (an antigen used for diagnosing Clostridium difficile infection). The circular DNA aptamer has higher stability, and the circular DNA aptamer is combined with rolling circle amplification to amplify signals more easily. Thus, the sequence of the region of the padlock probe I, III is responsible for the circularization of the padlock probe, and the region II sequence is designed to be a different sequence depending on the target but needs to avoid complementarity with the single-stranded target gene and formation of secondary structures with the regions I and III.

The invention designs another DNA oligo on the basis of Padlock technology, namely the invention is called as a blocker probe (a wild type blocker probe aiming at a wild type target gene and a mutant type blocker probe aiming at a mutant type target gene). The blocker probe is divided into two regions a and b, which are complementary pairs with the target DNA sequence (black), wherein the region a (gray) is the toehold region of the blocker (the region is prominent for binding with the target DNA sequence relative to the position where the blocker probe is bound with the target DNA sequence), and refer to FIG. 2. In addition, the padlock probe is a single-stranded DNA with a phosphate group at the 5' end, and can be divided into three regions I, II and III; where the I, III region is complementary paired to the target DNA sequence and the sequence of the I region (green) is the same sequence as the b region (green) of the blocker probe and the III region (purple) is the toehold region of the padlock probe (this region is prominent for binding to the target DNA sequence relative to the position where the blocker binds to the target DNA sequence). The action principle is as follows: firstly, the Blocker probe binds to the target DNA sequence, then the padlock probe preferentially binds to the target DNA sequence through the III region thereof, namely the toehold region, then the padlock probe replaces the Blocker from the target DNA sequence through strand displacement, and finally the T4 DNA ligase connects the padlock probe into a circular single-stranded DNA (namely cssDNA).

Through thermodynamic calculation, when designing the blocker probe and the padlock probe, the Gibbs free energy of the blocker probe bound to the target DNA sequence can be designed to be about equal to (the about equal to means that the Gibbs free energy of the blocker probe and the padlock probe are different by not more than 1-2Kcal/mol) the Gibbs free energy of the padlock bound to the target DNA sequence according to software such as NUPACK, so that the process of generating the cssDNA is a dynamic equilibrium state; when the kit is used for detecting single-site variation of nucleic acid, if a wild-type Blocker probe and a mutant DNA sequence (S) have a base mismatch, the Gibbs free energy of binding of the padlock probe to the target sequence is greater than that of binding of the wild-type Blocker probe to the target sequence with single-site variation, so that the binding of the padlock probe to the target sequence is more stable, and thus a product cssDNA is more formed, which is shown in electrophoresis that a cssDNA band is brighter than a control band without single-site variation, and a signal can be further amplified after the binding of the RCA, referring to FIG. 3; on the contrary, the same result as above is also observed when the mutant Blocker probe is mismatched with the wild-type DNA sequence (WT) by one nucleotide; however, this dynamic equilibrium state is maintained if the wild type/mutant Blocker probes correspond to the respective wild type/mutant DNA sequences.

In addition, in the Padlock probe-RCA technique, two Padlock probe sequences may hybridize to a target sequence at the same time, resulting in the formation of a polymer, as shown in FIG. 4; however, in the case of the action of the blocker probe, whether it is a wild-type template or a mutant template, the blocker probe preferentially binds to the target sequence, and then the Padlock probe displaces the blocker through the III region, i.e. the toehold region, since the same probe has a sequence (i.e. 5 'end) complementary to the target sequence, the 5' end of the same probe preferentially binds to the target, which reduces the generation of polymers and promotes the generation of the target css dna, as shown in fig. 5.

Preferably, the sequence length of the region a is 2-20nt, the sequence length of the region b and the sequence length of the region I are independently selected from 4-30nt, and the sequence length of the region III is 2-20 nt. The length of the II region sequence is 15-45 nt.

In some specific experiments, the invention verifies that the improved padlock probe + Blocker probe technology can avoid the problem of false negative or false positive through experiments, has higher specificity and sensitivity, reduces the generation of polymers, and can be applied to the detection of single-site variation of fragmented DNA, such as the detection of single-site variation of cfDNA by combining with the method for obtaining the single-strand pool provided by the invention.

In a specific comparison experiment, double-primer PCR based on a Lambda exonuclease degradation method is used as a control method (two pairs of primers with the span of 80nt and 150nt are respectively designed), the method for obtaining the single-strand pool disclosed by the invention and the method for obtaining the single-strand pool adopt the same cfDNA sample for amplification, and the result shows that on the premise of the same template, the method for obtaining the single-strand pool disclosed by the invention detects more mutant fragments than the method for obtaining the single-strand pool by using ordinary PCR, and the method has important significance for detecting the mutant. There are documents that show that ctDNA fragments are shorter than normal cfDNA fragments, and that the method of normal PCR using a two-primer system will lose those shorter fragments, the results of fig. 14 of the present invention confirm this conclusion, and the longer the span between two primers, the more fragments are lost, whereas the unbalanced PCR with only one primer of the present invention will see more complete mutant fragments.

According to the technical scheme, only one primer close to the site to be detected is used for carrying out PCR amplification on fragmented DNA under the condition that ddNTPs exist, a DNA single-chain pool with a short length and a ddNTP at the tail end is generated, the length of the amplified fragments is concentrated to 80-120nt, and compared with a method without the ddNTPs, the method enables the fragmented DNA to amplify short single-chain DNA fragments with the site to be detected more fully and comprehensively, the length of the generated fragments is shorter, and the length distribution is narrower. Meanwhile, when the Blocker-mediated padlock probe technology and other hybridization probes are subsequently applied to detection of fragmented DNA, the detection is more accurate and sensitive, and the method can be applied to relevant detection of fragmented DNA such as cfDNA.

Drawings

FIG. 1 is a schematic diagram showing the principle of the method for obtaining a single stranded cell according to the present invention;

FIG. 2 is a schematic view of the technical principle of the modified padlock probe + Blocker probe of the present invention;

FIG. 3 is a schematic diagram of the improved padlock + Blocker probe technique of the present invention for detecting single-site variations of nucleic acids; wherein S represents a target gene with single-site variation, and X represents a wild-type target gene;

FIG. 4 is a schematic diagram showing the principle of formation of multimers from padlock probes without a Blocker probe;

FIG. 5 is a schematic diagram showing the principle of lock probe type under a Blocker probe to avoid polymer production;

FIG. 6 is a schematic diagram of single-stranded DNA obtained by double-primer PCR based on Lambda exonuclease degradation;

FIG. 7 shows the results of capillary electrophoresis of single-stranded pool samples obtained by the method of the present invention and the double-primer PCR method based on Lambda exonuclease degradation; the upper panel shows the results of the present invention, and the lower panel shows the results of the double primer PCR method based on Lambda exonuclease degradation;

FIG. 8 shows the results of an assay to verify the reduction of polymer by the method of the invention; in the left panel, lane 1 is a linear padlock probe only; lane 2 is in the absence of the blocker probe, ligated with a linear padlock probe; lane 3 is a linear padlock probe ligation under the action of a blocker probe; bands at the position are the generated circular DNA products; in the right panel, lane 1 is a linear padlock probe only; lane 2 is in the absence of the blocker probe, ligated with a linear padlock probe; lanes 3-6 are ligated with a linear padlock probe under the action of the blocker3-6 probe;

FIG. 9 shows the results of detection of single-site variant nucleic acids using the modified padlock + Blocker probe technique of the invention; wherein, the left image is a gel image, and the right image is a quantification result of the gray value of the strip of the gel image; lane 1: add padlock probe in system (control); lane 2: no blocker is added, and the target is of a wild type sequence; lane 3: no blocker is added, and the target is a mutant sequence; lane 4: adding a wild type blocker, wherein the target is a wild type sequence; lane 5: adding a wild type blocker, wherein the target is a mutant sequence; q value is the gray value of the formed band with the target as the mutant sequence/the gray value of the formed band with the target as the wild type sequence; a is the Q value of no added blocker, B is the Q value of added wild type blocker;

FIG. 10 shows RCA results demonstrating the specificity and sensitivity of the improved padlock + Blocker probe technique of the invention in detecting single-site variant nucleic acids; the left graph is represented from top to bottom as a mixture of sequences containing 100%, 10%, 1%, 0.1% and 0% of the mutant sequences;

FIG. 11 is a schematic view of the detection principle;

FIG. 12 shows RCA detection results of cfDNA samples;

FIG. 13 shows RCA amplification curves of pure wild-type templates and pure mutant templates after detection according to the detection method of the present invention; wherein, none represents a No Blocker probe, two represent WT + SNV Blocker probes, and one represents a single wild-type WT Blocker probe;

FIG. 14 shows the results of comparing the method of the present invention with the Lambda exonuclease degradation based two-primer PCR method for preparing single-stranded DNA from the same sample for mutation type detection; s1 represents the D value obtained by mutant detection of single-stranded DNA prepared by the method used in the invention; PCR1 represents the D value obtained by mutant detection of 80nt single-stranded DNA prepared by combining traditional PCR with a Lambda exonuclease degradation method; PCR2 represents the D value obtained by mutant detection of 150nt single-stranded DNA prepared by combining traditional PCR with a Lambda exonuclease degradation method;

FIG. 15 is a graph showing the comparison of D values obtained by mutation detection of ssDNA prepared by the method of the present invention with and without ddNTPs.

Detailed Description

The invention discloses a method for obtaining a fragmented DNA single-chain pool and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content in the text for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method and its application have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention may be practiced and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit and scope of the invention.

The nucleic acid fragment, primer probe, reagent and the like used in the present invention can be synthesized and purchased by a reagent company.

The invention is further illustrated by the following examples.

Example 1: the invention discloses a method for obtaining a single-stranded pool and a double-primer PCR method based on a Lambda exonuclease degradation method

The schematic diagram of the double-primer PCR method based on the Lambda exonuclease degradation method is shown in FIG. 6;

in this example, a single complementary primer F1 of the invention was designed for cfDNA sample mutation sites, and paired primers R1 and R2 with lengths of 80nt and 150nt, respectively, between F1 were designed to form two sets of paired primers F1 and R1, and F1 and R2; see table 1 for details;

TABLE 1

cfDNA samples: EGFR mulltiplex cfDNA Reference Standard (cfDNA containing 1% EGFR L861Q mutation, commercially available);

wild-type sequence:

ACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACC；

mutant sequence:

ACAGATTTTGGGCTGGCCAAACAGCTGGGTGCGGAAGAGAAAGAATACC；

the oligonucleotide fragments used in Table 1 above were all purchased from Suzhou Jinwei Zhi Biotech Co., Ltd (F1 sequence shown in SEQ ID NO: 14)

The method for obtaining the single-chain pool comprises the following steps:

the cfDNA sample is phosphorylated at the 5' end by using T4 phosphokinase, and then single primer F1 and a mixture of nucleic acid monomers dNTPs and ddNTPs in a certain proportion are added for PCR amplification (other components of a PCR amplification system are referred to the conventional method, and the details are shown in Table 2). Degrading the template by using lambda exonuclease after the amplification is finished to obtain a cfDNA single-chain pool to be detected;

a double-primer PCR method based on a Lambda exonuclease degradation method comprises the following steps:

phosphorylation of the 5' end of the same cfDNA sample using T4 phosphokinase followed by PCR amplification with the addition of paired primers F1 and R1 or F1 and R2 (no ddNTPs added, other PCR amplification systems are the same as the present invention, see Table 3 for details); degrading the template by using lambda exonuclease after the amplification is finished to obtain a cfDNA single-chain pool to be detected;

TABLE 2

TABLE 3

component	50uL	Final Conc.
			H₂O	28.5	--
5x Phusion reaction buffer	10	1x
			dNTPs(2.50M)	4	0.2mM
F1(100uM)	2	4uM
			R1/R2(100uM)	2	4uM
1％EGFR(PO₄-)(10ng/uL)	3
			Phusion	0.5

PCR procedure:

1.98℃2min

2.98℃30s

3.54℃30s

4.72℃5s

procedures 2-4 were repeated for 150 cycles

An exonuclease digestion reaction system:

TABLE 4

component	30uL	Final Conc.
			H₂O	1
10x lambda buffer	3	1x
			PCR product	25
Lambda exonuclease	1

The exonuclease digestion reaction condition is as follows:

the Lambda exonuclease was inactivated at 37 ℃ for 3h and then at 75 ℃ for 10 min.

Example 2: feasibility verification of the improved padlock probe + Blocker probe technology

In this embodiment, the sequences in table 5 are selected for feasibility analysis, but this should not be a limitation of the present invention, and different probe sequences can be designed for targets by software according to the principles and schemes provided by the present invention, which is not exhaustive;

TABLE 5

Note: table 5 all Blocker probes are wild-type probes;

in Table 5, the sequence of the Padlock probe (Padlock probe) is the I region sequence in italic part, the III region sequence (free energy of-17.49 Kcal/mol) in underlined part, and the II region sequence in the rest; the wild type template and the mutant template are G-T mutation of 16 th base; the italic part of the sequence of the Blocker 1 is a b region sequence, and the other part is an a region sequence (the free energy is-17.04 Kcal/mol); the italic part in the Blocker 2 sequence is a b region sequence, namely no a region sequence is 0, and the free energy is 0 correspondingly;

in the sequence of the Blocker3-6, the italic part is a b region sequence, and the other is an a region sequence (the free energy is-21.99 Kcal/mol, -18.7Kcal/mol, -23.43Kcal/mol and-18.7 Kcal/mol in sequence);

according to Gibbs free energy, Padlockprobe + Blocker 1 can be used for detecting single-site variation, and Padlockprobe + Blocker 2 is used for generating single-stranded circular DNA (promoting the generation of more single-stranded circular DNA), and both can inhibit the generation of polymer;

the oligonucleotide fragments used in Table 5 above were purchased from Yuzhi Biotech, Suzhou (SEQ ID NO:1-9 in sequence), and were modified by phosphorylation at the 5' end of the padlock probe. T4 DNA ligase, Exo I, Exo III, Bst DNA polymerase were purchased from New England Biolabs (NEB). dNTPs were purchased from Beijing Quanjin Biotechnology Ltd. SYBR Gold Nucleic Acid Gel Stain was purchased from Saimer fly. SYBR Green I was purchased from Solebao Biotech, Inc. of Beijing.

The polyacrylamide gel electrophoresis adopted in the invention is 15% modified polyacrylamide gel. The gel system is shown in Table 6:

TABLE 6

The 300V voltage is firstly subjected to pre-electrophoresis for 30min, then the sample is loaded, and the 120V voltage is subjected to electrophoresis for 3 h. SYBRGold Nucleic Acid Stain was then stained for 20min and imaged in a gel imager (Azure C300).

1. Test to verify reduction of Polymer

The following table 7 system for connection;

TABLE 7

The above ligation system was incubated in a PCR instrument (Eppendorf Mastercycler) at 37 ℃ for 3h, after which T4 DNA ligase was inactivated at 65 ℃ for 10 min. Then 2.5UExo I and 10U Exo III were added, incubated at 37 ℃ for 3h, and Exo I and Exo III were inactivated at 80 ℃ for 20 min. Finally, polyacrylamide gel electrophoresis was performed, and the results are shown in FIG. 8.

Lane 1 on the left panel of figure 8 is a linear padlock probe only; lane 2 shows the formation of polymers (polymers) by ligation using a linear padlock probe without the action of a blocker probe, at the position of the circular DNA product; lane 3 shows ligation with a linear padlock probe under the action of a blocker probe, and it can be seen that the formation of multimers is greatly reduced or even absent, and the amount of cyclic products is increased, indicating that ligation efficiency is not affected.

The right graph is the verification condition by using other block probes, the result is consistent with the left graph, and the generation of the polymer is greatly reduced compared with the generation of a2 nd lane without adding the block probes, and the amount of the cyclic product is improved, which shows that the connection efficiency is not influenced.

2. Detection of Single base variations

The following table 8 was used for the ligation;

TABLE 8

The above ligation system was incubated in a PCR instrument (Eppendorf Mastercycler) at 37 ℃ for 3h, after which T4 DNA ligase was inactivated at 65 ℃ for 10 min. Then 2.5UExo I and 10U Exo III are added, the mixture is incubated at 37 ℃ for 3h and at 80 ℃ for 20min to inactivate the Exo I and Exo III (in the process of generating the cssDNA, a looped padlock probe and a linear padlock probe may exist at the same time, and in order to avoid the influence of the linear padlock probe on the cssDNA during electrophoresis detection, the characteristic that the single-stranded DNA is hydrolyzed by exonuclease specificity instead of the circular single-stranded DNA is generally utilized, so the exonuclease I and the exonuclease III are used). Finally, polyacrylamide gel electrophoresis was performed, and the band gray values were quantified, as shown in FIG. 9.

In FIG. 9, lane 1 shows the padlock probe added to the system only (control); lane 2 is without blocker, target wild type sequence; lane 3 without blocker, target mutant sequence; lane 4 is with a wild type blocker added, the target is a wild type sequence; lane 5 is the addition of a wild type blocker, targeting the mutant sequence. As can be seen from the figure, in the absence of the blocker, the efficiencies of the wild-type template and the mutant template for padlock looping are consistent (Q value is about 1); however, in the case of addition of wild-type Blocker, the loop formation of the mutant template was 16 times that of the wild-type template. This also illustrates that the blocker in the improved technique of the present invention is covering for fully complementary sequences; but the effect of binding the template is weakened for the sequences with base differences, which exactly verifies the principle described in the attached figure 3; this is of great interest for future clinical applications. Because the majority of clinical samples are wild-type sequences and few mutant sequences exist, the signals of the wild-type sequences are far greater than those of the mutant sequences in the current nucleic acid detection technology, and false negative results. The same results can also be expected if the relevant experiment is performed by replacing it with a mutant Blocker.

The band marked by the symbol in lanes 2-5 is where the circular product is located, the band at the same position as the lane 1 control is due to complete degradation of exonuclease I/III.

3. Detection of specificity and sensitivity

To verify the sensitivity and specificity of the improved technique for the detection of single base variations, this example performed 0.1% of experiments, i.e., the detection of 1 mutant sequence out of 999 wild-type sequences. That is, the mutant sequence and the wild type sequence are mixed in different proportions, which are 0%, 0.1%, 1%, 10% and 100%, respectively. Then, the target sequence is used for detection, amplification signals are collected through connection, exonuclease degradation and real-time fluorescent rolling circle amplification, and a connection system is shown in a table 9;

TABLE 9

The above ligation system was incubated in a PCR instrument (Eppendorf Mastercycler) at 37 ℃ for 3h, after which T4 DNA ligase was inactivated at 65 ℃ for 10 min. Then 2.5UExo I and 10U Exo III were added, incubated at 37 ℃ for 3h, and Exo I and Exo III were inactivated at 80 ℃ for 20 min. Finally, real-time fluorescent rolling circle amplification is carried out, and an RCA amplification system is shown in a table 10;

watch 10

The amplification mixture was incubated at 55 ℃ for 1h in QuantStaudio 6Flex Real-Time PCR Systems and fluorescence signals were collected every 30s, and the results are shown in FIG. 10.

As can be seen from the results in FIG. 10, the improved technique of the present invention achieves the detection of 0.1% mutant sequences, indicating that the improved technique of the present invention has higher specificity and sensitivity for the detection of single base variations.

Example 3: validation of method for detecting cfDNA

On the basis of the embodiment 1, the improved padlock probe and Blocker probe technology is combined to detect an EGFRUltiplex cfDNA Reference Standard sample, the designed related probes are shown in the following table 8, a connection system for adding different Blocker probes is shown in tables 11-14, and an RCA reaction system is shown in the following table 15;

TABLE 11

In Table 11, the sequence of the Padlock probe (Padlock probe) is the I region sequence in italic part, the III region sequence (free energy of-19.38 Kcal/mol) in underlined part, and the II region sequence in the rest; the wild type template and the mutant template are G-T mutation of the 23 rd base; the italic part of the sequence of the Blocker 1 is a b region sequence, and the other part is an a region sequence (the free energy is-19.24 Kcal/mol); the italic part of the sequence of the Blocker 2 is a b region sequence, and the other part is an a region sequence (the free energy is-19.63 Kcal/mol);

the oligonucleotide fragments used in Table 11 above were all purchased from Suzhou Jinwei Zhi Biotech Co., Ltd (SEQ ID NO:10-13 in the order of sequence)

TABLE 12 double Blocker (wild + mutant) ligation systems

TABLE 13 Single Blocker (wild) ligation systems

TABLE 14 Blocker-less connection hierarchy

The connection reaction conditions are as follows:

37℃3h,then 65℃10min to inactive T4 DNA ligase

TABLE 15RCA reaction System

As shown in the detection principle of fig. 11 and the specific RCA result of fig. 12, a3 is a Ct value (Ct ═ 96.65) obtained by RCA of the ligation product in the case of adding the double blokcer, which represents the generation of Ct from the background; a2 is the Ct value obtained for RCA of the ligation product with wild-type Blocker addition (Ct ═ 65.03), which represents the Ct resulting from (mutation + background); a1 is the Ct value obtained by RCA of the ligation product in the absence of the blocker, which represents the Ct (Ct 42.97) generated in the system (total analytes + background). (A3-A1) is the Ct produced by the total analyte to be detected; (A3-A2) is the Ct produced by the mutation; (A3-A2)/(A3-A1) is the ratio of mutations calculated from the experiment and is the D value. In FIG. 12, if A2 is close to A3, the mutation rate is low, and close to A1, the mutation rate is high, and the higher the D value is, the higher the mutation rate is.

In order to verify that the difference between the Ct values obtained under the reaction conditions of the double-blocker and the single-blocker is not caused by adding one more blocker but caused by the existence of the mutant template, the RCA amplification is performed by using the pure wild-type template and the pure mutant template according to the test method of the embodiment, and the amplification curve chart is shown in fig. 13;

FIG. 13 shows that when the frequency of the mutant gene is 100% (pure mutant template), the amplification curves with wild-type Blocker alone and without any Blocker are substantially overlapped, indicating that wild-type Blkcer does not block the mutant template by one-base difference, and the detection result is substantially the same as that without Blocker because the padlock probe is not involved in the complementation of the mutation site. When the frequency of the mutant gene is 0% (pure wild-type template), amplification curves of adding double blocks and adding only one block are basically overlapped, so that the situation that the mutant-type block has one base difference and does not have the effect of closing the wild-type template is shown, and the wild-type block exists in the two amplification systems and can be complemented with the pure wild-type template for closing, so that the detection results are basically the same.

Example 4: the method for obtaining the single-stranded pool and the RCA detection contrast of the method for obtaining the single-stranded pool by the double-primer PCR based on the Lambda exonuclease degradation method

The single strand pool products prepared by the two methods were obtained as described in example 1, then examined according to the method of example 3 and the D values were calculated in accordance with the formula (A3-A2)/(A3-A1) in unison, and the results are shown in FIG. 14;

it is obvious from fig. 14 that, under the same template, the method for obtaining single-stranded pool of the present invention detects more mutant fragments than the method for obtaining single-stranded pool of the general PCR, which is of great significance for detecting mutant. There is literature that ctDNA fragments are shorter than normal cfDNA fragments, and the method using the two-primer system normal PCR will lose those shorter fragments, fig. 14 corroborates this conclusion, and the longer the span between the two primers, the more fragments are lost, whereas the unbalanced PCR with only one primer of the present invention will see more complete mutant fragments.

Example 5: effect of ddNTPs on the method of obtaining Single Strand cell of the present invention

1. DNA Single Strand Length Effect

Referring to the method of obtaining a single-stranded cell according to the present invention described in example 1, the single-stranded cell was prepared by adding ddNTPs and dNTPs (24:1), and adding only dNTPs as two test groups, and the single-stranded cells obtained from the two test groups were sent to a reagent detection company for capillary electrophoresis analysis (Qsep 100DNA fragment analyzer), and the results are shown in FIG. 7. As can be seen from FIG. 7, most of the fragment lengths in the single-stranded pool of cfDNA generated by the test group without adding ddNTPs are concentrated between 100nt and 200nt, most of the fragment lengths in the single-stranded pool of cfDNA generated by adding ddNTPs are concentrated between 80nt and 120nt, the fragments are obviously shortened, the probability of forming a secondary structure of the generated single-strands is greatly avoided, and the detection sensitivity is improved.

2. Mutation rate detection impact

The single-stranded cells were prepared by the method of obtaining single-stranded cells according to the present invention described in example 1, with the addition of ddNTPs and dNTPs (1:50), and with the addition of dNTPs alone as two test groups, and were examined and the D value was calculated in accordance with the formula (A3-A2)/(A3-A1) in a unified manner, as described in example 3, and the results are shown in FIG. 15;

as is obvious from 15, if no ddNTPs is added, a great number of mutation samples are missing in the obtained single-chain pool, so that the real mutation rate cannot be presented in the subsequent detection; the mutation rate detected by the invention is obviously higher than that of a test group only added with dNTPs, so that richer mutation conditions can be presented, and the loss is avoided.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Tianjin university

<120> method for obtaining fragmented DNA single-strand pool and application thereof

<130> MP2023196

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 51

<212> DNA