CN106282353B - Method for carrying out multiple PCR by utilizing hairpin primer - Google Patents

Abstract

The invention relates to a method and a kit for performing multiplex PCR by utilizing hairpin primers, and application in establishing a second-generation sequencing platform library. The hairpin primer 3' end specific sequence is completely matched with the target segment, the melting temperature is above 80 ℃, and the hairpin structure is only opened in a denaturation procedure during PCR reaction. Can effectively reduce non-specific amplification and formation of primer dimer, and improve the uniformity of multiple PCR products. The invention also utilizes multiple hairpin primers to carry out two-round PCR reaction to quickly and conveniently construct a second-generation sequencing library, and distinguishes different templates through the adapter primers carrying different barcode sequences, and the different adapter primers carry the same universal amplification primer so as to reduce the difference of amplification products of different templates.

Description

Method for carrying out multiple PCR by utilizing hairpin primer

Technical Field

The invention relates to the field of nucleic acid detection, in particular to a multiplex PCR technology which utilizes hairpin primers to carry out multiplex PCR, enriches DNA sequences of a plurality of sample target sections and reduces primer dimer and non-specific amplification.

Background

Multiplex PCR is an important means of enrichment technology for target segments due to its low cost and simple operation. In the face of target segment capture of a large number of complex genome samples, the multiplex PCR technology has become the first choice technology due to the advantages of strong specificity, low price, good repeatability and the like. Meanwhile, commercial multiplex PCR kits and non-commercial multiplex PCR technologies are emerging. According to the literature "Target-variant sequences for next-generation sequencing" (Naturemethods,2010, 7(2):111-118) and "Genotyping-in-Thousan by sequencing (GT-seq): A cost effective SNP sequencing method based on stored amplification sequencing" (Molecular analysis resources,2015,15(4):855-867), suppliers such as Thermo Fisherscientific and illumina provide commercial multiplex PCR kits for tumor detection; non-commercial techniques also include long-fragment PCR, microdroplet PCR, patch PCR, and the like.

Compared with other target section enrichment technologies, the multiplex PCR has strong specificity and low price, and does not need expensive hardware facilities. However, the throughput of conventional multiplex PCR is limited by the number of primer pairs. In conventional multiplex PCR reactions, primer mismatches and non-specific amplification products increase dramatically as the primer pair number increases. The interaction between a plurality of pairs of primers not only introduces a large amount of non-specific amplification products, but also causes great difference of copy numbers of different target fragments; therefore, conventional multiplex PCR is generally limited to amplification of 10-20 primers, and uniform products cannot be obtained beyond this limit. How to optimize the reaction conditions of multiplex PCR, and reducing primer dimer, primer mismatch and non-specific amplification are key factors for increasing the throughput of multiplex PCR, some multiplex PCR optimization strategies have been reported in recent years, but still need to be further improved.

Disclosure of Invention

Technical problem to be solved

The technical problem to be solved by the invention is to provide a method for performing multiplex PCR by utilizing hairpin primers, which improves the flux of the multiplex PCR and overcomes the defect of non-uniformity of target products caused by mismatching and non-specific amplification of a plurality of pairs of primers in the traditional multiplex PCR technology.

Technical scheme

In a first aspect of the invention, there is provided a method of multiplex PCR using hairpin primers, wherein the primers which specifically pair to the target segment sequences are hairpin primers. Preferably, the 3 ' end of the hairpin primer is specifically paired with the target segment sequence and the 5 ' end of the hairpin primer is paired with the 3 ' end sequence. More preferably, the base at the 3 'end of the hairpin primer is complementary paired with the base at the 5' end, such that the 5 'end and the 3' end of the hairpin primer are blocked.

as used herein, a "hairpin primer" refers to a primer having a "hairpin structure" or "stem-loop structure". In a preferred embodiment, the hairpin primer has a target segment-specific sequence of about 20bp at the 3 ' end, and the 5 ' end sequence is complementary-paired with the 3 ' end sequence to form a stem. The rest part is a ring part. A hairpin primer refers to a pair of primers whose upstream and downstream are hairpin structures, unless otherwise specified. Preferably, the number of the upstream and downstream hairpin primers in the same PCR reaction is two or more pairs.

in a preferred embodiment of the present invention, the hairpin primer has a melting temperature of not lower than 80 ℃ and lower than the denaturation temperature of multiplex PCR. Preferably, the hairpin primer has a melting temperature of 81 ℃ to 86 ℃. At normal temperature or below the melting temperature, the hairpin primer will form a stem-loop structure itself, and at the denaturation temperature in the PCR reaction, the hairpin structure in which the 5 'end sequence and the 3' end sequence of the stem-loop structure hybridize in a matched manner is opened. The inventors have unexpectedly discovered that performing multiple amplification reactions when the hairpin primer 3' specific sequence is fully paired with the target segment can greatly reduce non-specific amplification and primer dimer production.

In another preferred embodiment of the above method, the loop of the hairpin primer other than the sequence corresponding to the 5 'end and the 3' end comprises a universal sequence. The universal sequence, as a specific embodiment of the present invention, is, for example, a non-human gene sequence when the target sequence is a human gene.

In a second aspect, the present invention provides a multiplex PCR kit based on the above method, comprising a plurality of pairs of said hairpin primers.

The third aspect of the present invention provides a method for constructing a second-generation sequencing platform library by using hairpin primers, comprising the following steps:

(1) Respectively adding an upstream universal sequence and a downstream universal sequence at the 5 ' ends of the upstream and downstream of a specific primer sequence designed aiming at a target segment, and respectively adding sequences which are completely complementary and paired with the 3 ' end part sequence of the target segment at the 5 ' ends of the universal sequences, so that the melting temperature is not lower than 80 ℃, thereby obtaining an upstream hairpin primer and a downstream hairpin primer with closed ends;

(2) A first round of PCR reaction, wherein two or more pairs of upstream and downstream hairpin primers are used for amplifying a target segment for 10-30 cycles, preferably 15-25 cycles;

(3) Designing an adapter primer, which comprises three sequences, namely a second-generation sequencing adapter primer, a bar code (namely barcode) sequence and a universal sequence from the 5' end; wherein the barcode sequence of each pair of adapter primers is different to distinguish different templates;

(4) And (3) taking stock solution or diluent of the first round of PCR amplification products as a template, adding upstream and downstream joint primers to perform second round PCR reaction, and mixing second round amplification products obtained from different samples to form a second-generation sequencing platform library.

the fourth aspect of the invention provides a kit for constructing a second generation sequencing platform library based on the method, which comprises a plurality of pairs of the hairpin primers and the adapter primers.

Further, the specific manner adopted by the above technical solution may include, for example, the following steps:

Designing a hairpin primer: designing a Primer aiming at a target segment sequence to be sequenced, designing by using Primer3 online software (http:// frodo.wi.mit.edu/Primer3/) according to a sequence in an NCBI database, and manually adding a pairing sequence which is completely complementary with a 3 'end part sequence of the target segment at the upstream and downstream 5' ends of the designed Primer to form a hairpin Primer with a completely closed end, and simultaneously ensuring that the melting temperature of the hairpin Primer is not lower than 80 ℃. It should be noted that for the third aspect of the invention: when the hairpin primer is used for constructing a second-generation sequencing platform library, a universal sequence can be added at the 5 ' end of a target section, and then a complete complementary pairing sequence with the 3 ' end part sequence of the target section is added at the 5 ' end of the universal sequence to obtain a specific hairpin primer with a completely closed end. It should be understood that whether to add a universal sequence to the 3' end of the target segment does not limit the present invention.

for example, a PCR amplification system is that 25ng of DNA is added into 30 muL of Taq enzyme system, wherein the Taq enzyme system comprises 0.01 muM of each pair of specific primers, 200 muM dNTPs, 0.2mM MgCl ₂, 1X PCR Buffer, 1.2U Taq enzyme, and mineral oil is used for covering, and a negative control is set at the same time, the reaction procedure is that 95 ℃ denaturation is carried out for 3min, 94 ℃ is carried out for 30s, 60 ℃ renaturation is carried out for 4min, and 28 cycles.

The inventors tested the number of pairs of primers allowed by the first round of multiplex PCR, i.e., the "weight" number of multiplex PCR reaction, through multiple experiments, and found that under 20 (i.e., 20 pairs of primers are amplified in one reaction system at the same time), 50-60, and at most 89, a uniform platform library suitable for second-generation sequencing can be obtained, and expected technical effects can be obtained. Although no more multiplex PCR experiments were performed, the inventors believe that 100, even 150, by weight using the method of the invention may be successful in achieving the inventive concept. More multiplex reactions may require preference to the reaction system or reaction conditions of multiplex PCR, but should still be a preferred way within the technical solution of the present invention. Meanwhile, the multiple PCR is not the decision or the only factor influencing the construction of the final platform library and the uniformity thereof, so the invention is not limited by the conception, and the multiple can be selected according to the actual requirement in the process of implementing the invention.

In the experiment, the inventor finds that if the hairpin primer design of the invention is omitted and the traditional two-round PCR amplification is directly carried out, more than 20 pairs of primers exist in the reaction system, a large amount of primer dimers or mismatching can not be avoided, so that no obvious target band appears, and the product can not be used as a second-generation sequencing library. Therefore, in the art, multiplex PCR is generally limited to amplification of 10-20 primer pairs, and beyond this limit, the desired product cannot be obtained.

Advantageous effects

The present invention provides a protocol for the enrichment of multiple target segments using hairpin primers in conjunction with multiplex PCR that is lacking in the prior art literature. During PCR amplification, multiple PCR is carried out by utilizing the hairpin primers, so that the formation of primer dimers and non-specific amplification among a plurality of pairs of primers is reduced, and the uniformity of different amplicons is improved; meanwhile, the number of the amplified primer pairs is far more than that of the common multiplex PCR, and the efficiency and the flux of the multiplex PCR are improved. The multiple PCR scheme of the invention realizes the capability of parallel capture aiming at dozens of samples, greatly saves the cost and effectively improves the capture efficiency.

the invention and its effects are further explained below with reference to the drawings and the examples.

Drawings

FIG. 1 is a schematic diagram of a primer of the present invention. In the figure, 101 is an upstream adaptor primer, 102 is a downstream adaptor primer, 103 is an upstream specific hairpin primer, and 104 is a downstream specific hairpin primer.

FIG. 2 is a schematic diagram of a specific hairpin primer of the invention. In the figure, 201, an upstream specific hairpin primer, 202, a downstream specific hairpin primer, 203, an upstream universal sequence, 204, a downstream universal sequence, 205, a sequence that is perfectly complementary to the sequence of the 3 'end portion of the target segment of the upstream specific primer, 206, a sequence that is perfectly complementary to the sequence of the 3' end portion of the target segment of the downstream specific primer, 207, a sequence of the upstream specific primer, 208, a sequence of the downstream specific primer.

FIG. 3 is a schematic diagram of an adapter primer of the present invention. In the figure, 301. upstream adaptor primer, 302. downstream adaptor primer, 303. next generation sequencing upstream adaptor primer, 304. next generation sequencing downstream adaptor primer, 305. upstream adaptor primer barcode sequence, 306. downstream adaptor primer barcode sequence, 307. universal primer upstream sequence, 308. universal primer downstream sequence, 309. specific hairpin primer amplification product.

FIG. 4 is a distribution diagram of effective amplicons when detecting single nucleotide polymorphisms using the Ion torrent PGM platform according to the present invention. In the figure, 401 is the number of amplicons, 402 is the depth of sequencing of the amplicons.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

the following examples are examples of experimental procedures not specifically indicated, generally according to conventional conditions, such as the manual of molecular cloning operations, or according to the conditions recommended by the manufacturers of reagents all inorganic chemical reagents and organic solvents were purchased from Shanghai chemical reagents, Inc., Taq DNA polymerase, dNTPs were purchased from Promega, USA, primers were synthesized by Shanghai Biotech, rat DNA extraction Using the Rapid extraction Kit for animal genomes from Biotechnology, Inc. (Shanghai), agarose gel recovery Sequencing library was obtained using the recovery Kit from Tiangen Biotechnology, Inc., Sequencing Kit IonPGM ^TM Sequencing 200Kit v2 was purchased from Thermo Fisher Scientific.

Example 1:

Single Nucleotide Polymorphisms (SNPs) were detected using the Ion torrent PGM platform.

Sequence search and primer design: according to the SNP database of NCBI, a total of 89 target segment sequences carrying SNP sites in a rat genome are found out, Primer design is carried out by using Primer3 online software according to a general rule, upstream and downstream universal sequences are respectively and manually added at the upstream and downstream 5 ' ends of the designed Primer, a sequence which is completely complementary and matched with about 13bp of the 3 ' end of the target segment is added at the 5 ' end of the universal sequence to form a hairpin Primer with a completely closed tail end, and the melting temperature of the hairpin Primer is ensured to be not lower than 80 ℃, and then Primer synthesis is carried out (Table 1).

TABLE 1 specific hairpin primers for SNP detection

DNA extraction: the 44 groups of rat DNA are extracted by adopting an animal genome rapid extraction kit according to the instruction to obtain DNA, and the quality and the concentration of the DNA are determined by 0.8% agarose gel electrophoresis. The correspondence between 44 sets of DNA and the adapter primer is shown in Table 2. The 44 samples differed in adaptor primer to distinguish between the different template libraries (Table 3).

TABLE 2 sample number and adapter primer correspondence table

TABLE 3 adaptor primer sequence Listing

Two rounds of PCR amplification were performed on 44 sets of sample DNA.

In the first round of PCR, all 89 pairs of upstream and downstream specific primers in the table 1 are selected and mixed in the same reaction system as amplification primers, 89-fold PCR reaction is carried out on a single sample, wherein the PCR amplification system specifically comprises that 25ng of DNA of each sample is respectively added into a 30 mu L Taq enzyme system, the Taq enzyme system comprises 0.01 mu M of each pair of specific primers, 200 mu M dNTPs, 2mMMgCl ₂, 1X PCR Buffer and 1.2U Taq enzyme and is covered by mineral oil, and simultaneously, a negative control is set, the reaction program comprises the steps of denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, renaturation at 60 ℃ for 4min and 22 cycles.

The second round of PCR selects the first round of PCR products as templates, and the PCR amplification system is characterized in that 1 mu L of the first round of PCR products are respectively added into respective second round of 10 mu L of Taq enzyme systems, wherein the Taq enzyme systems comprise 1 mu M upstream and downstream adapter primers, 200 mu M dNTPs, 2mM MgCl ₂, 1X PCRbuffer and 0.4U Taq enzyme and are covered by mineral oil, and the reaction procedures are 95 ℃ denaturation for 3min, 94 ℃ denaturation for 30s, 65 ℃ renaturation for 2min, 72 ℃ extension for 30s and 10 cycles.

After amplification is finished, amplification products of 44 samples are uniformly mixed together in equal volume to serve as a constructed Sequencing library, Ion torrent PGM platform Sequencing and typing are simultaneously carried out on the 44 samples, the Sequencing library is purified according to the specification of an agarose gel recovery Kit, the purified library is subjected to subsequent emulsion PCR and enrichment according to a PGM commercial operation flow, the enriched products are subjected to secondary Sequencing by using 318 chips and Ion PGM ^TM Sequencing 200Kit v2, and the steps are strictly used according to the requirements of suppliers.

The sequencing results are shown in FIG. 4. The uniformity of multiplex PCR was judged by detecting the number of measured sequences (reads) of the different amplicons.

Sequencing of all amplicons on PGM in 44 samples showed that 99.6% of the amplicons had at least 1 determined sequence number (reads), while 87.8% had sequences greater than or equal to 20 and less than 5000. The difference between the effective amplicons (. gtoreq.20 and <5000reads) detected in this example was 1-2 orders of magnitude. Figure 4 results show that: the effective amplicon differences of 72.8%, 90.1%, 97.6%, 99.2% and 99.7% were within 25-fold, 50-fold, 100-fold, 150-fold and 200-fold, respectively. The uniformity of multiplex PCR using hairpin primers was comparable to that of some published multiplex PCR Methods in the background art, in comparison with the data reported in references "Methods for genomic partitioning" (Annual review of Genetics & Human Genetics,2009,10:263-284) and "A high-plex PCR for mapping multiplex sequencing" (Biotechniques,2013,55(2):69-74) (Table 4).

The results in table 2 show that not only the uniformity difference for each amplicon is reduced, but the uniformity difference for each sample is also reduced. Of the 44 samples, 100% of the samples differed by 27-fold or more, ensuring sufficient sequencing throughput for each sample.

TABLE 4 comparison of homogeneity for different amplification protocols

Amplification method	Uniformity of
		Hairpin primer multiplex PCR of example 1	99.7% of the amplicons were within 200-fold difference
High-plexPCR	98.33% is within 100 times
		intramolecular probe	58% is within 10 times, 88% is within 100 times
Patch PCR	75% is within 50 times
		liquid hybridization	80% is within 25 times

Claims (5)

1. A non-disease diagnosis and treatment method using hairpin primer to do multiplex PCR is characterized in that the primer specifically matched with the target segment sequence is hairpin primer, the 3 ' end of the hairpin primer is specifically matched with the target segment sequence, the 5 ' end of the hairpin primer is matched with the 3 ' end sequence, the melting temperature of the hairpin primer is not lower than 80 ℃ and lower than the denaturation temperature of multiplex PCR, the ring part of the hairpin primer except the matching sequence of the 5 ' end and the 3 ' end is universal sequence, the number of the upstream hairpin primer and the downstream hairpin primer in the same PCR reaction is two or more, the base of the 3 ' end of the hairpin primer is complementary matched with the base of the 5 ' end, so as to seal the 5 ' end and the 3 ' end of the hairpin primer.

2. A kit for the method of claim 1, comprising a plurality of pairs of said hairpin primers.

3. A non-disease diagnosis and treatment method for constructing a second-generation sequencing platform library by utilizing hairpin primers comprises the following steps:

(1) respectively adding an upstream universal sequence and a downstream universal sequence at the 5 ' ends of the upstream and downstream of a specific primer sequence designed aiming at a target segment, and respectively adding sequences which are completely complementary and paired with the 3 ' end part sequence of the target segment at the 5 ' ends of the universal sequences, so that the melting temperature is not lower than 80 ℃, thereby obtaining an upstream hairpin primer and a downstream hairpin primer with closed ends;

(2) Performing a first round of PCR reaction, namely performing 10-30 cycles of amplification on a target section by utilizing a plurality of pairs of upstream and downstream hairpin primers;

(3) Designing an adaptor primer, which comprises three sequences, namely a second-generation sequencing adaptor primer, a bar code sequence and a universal sequence from the 5' end; wherein the barcode sequence of each pair of adapter primers is different to distinguish different templates;

(4) And (3) taking stock solution or diluent of the first round of PCR amplification products as a template, adding upstream and downstream joint primers to perform second round PCR reaction, and mixing second round amplification products obtained from different samples to form a second-generation sequencing platform library.

4. The method of claim 3, wherein the first round of PCR is performed for 15-25 cycles.

5. A kit for the method of claim 3, comprising a plurality of said pairs of hairpin primers and adapter primers.