CN113166801A

CN113166801A - PCR primer, PCR amplification method and application

Info

Publication number: CN113166801A
Application number: CN201880099626.9A
Authority: CN
Inventors: 杨林; 杨贵芳; 张艳艳; 陈芳; 蒋慧
Original assignee: MGI Tech Co Ltd
Current assignee: MGI Tech Co Ltd
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-07-23
Also published as: WO2020124472A1

Abstract

A PCR primer, a PCR amplification method and application thereof, wherein a forward primer and a reverse primer in the primer both comprise a same fixed sequence, and the forward primer and the reverse primer respectively comprise a specific primer sequence different from each other. The primer of the invention enables the primer dimer to form a neck ring structure by itself through fixing the sequence to inhibit the primer dimer from being used as a template for subsequent PCR amplification, thereby greatly reducing the primer dimer in the PCR process.

Description

PCR primer, PCR amplification method and application

Technical Field

The invention relates to the technical field of PCR amplification, in particular to a PCR primer, a PCR amplification method and application.

Background

In the post-genomic era, there is an increasing need for re-sequencing candidate segments of the genome, with sequence concerns over a few SNPs, and candidate segments may range from 5Kb to 10M. The use of traditional sanger methods or whole genome sequencing is expensive and the use of target region capture sequencing solves this problem well. Target area capture techniques can be broadly divided into two categories: one based on hybridization capture sequencing technology, another based on multiple PCR capture technology. The two are used for capturing the interested gene region at one time through a plurality of probes or primers, and a high-throughput sequencing technology is combined, so that a plurality of samples are sequenced simultaneously to obtain the sequence information of the target region. The target capture sequencing technology greatly reduces cost while screening large sample volumes relative to whole genome sequencing. However, the former has complicated experimental procedures and higher probe cost, limits the clinical application of the probe, and the latter has simple experimental operation and strong flexibility, and is suitable for screening and diagnosis of Mendelian hereditary diseases, re-sequencing of GWAS candidate segments, re-sequencing of QTL positioning segments, and precise medical research and application.

The high-throughput SNP detection service combines a multiplex PCR and a high-throughput sequencing technology, a specific primer is designed for a site needing to be detected, multiplex PCR amplification is carried out in a single tube, and different samples are distinguished by different tag (barcode) primers. After the samples are mixed, the amplicons are sequenced on a sequencing platform, different samples are distinguished by using a bioinformatics method as a sequencing result, and finally the SNP information of each site is obtained. The method is suitable for different purposes of genetic research, such as disease genome research, tumor genome research, disease and gene association research, clinical molecular diagnosis and the like, can be used for QTL positioning and molecular breeding in plant genome research, and is very suitable for SNP analysis of large-scale samples. Although the experimental operation is simple and the cost of single detection is low, the method needs to repeatedly test and optimize a plurality of pairs of primers in the early stage of the experiment, and wastes time and labor. Especially in ultra-heavy PCR, the complexity of the primer sequence makes it easy for the primers to form primer dimers. The formation of primer dimer will rapidly consume the raw materials in the PCR reaction system, resulting in that PCR quickly reaches the plateau phase; the formed primer dimer can be sequenced in subsequent sequencing, invalid data is formed, and the utilization efficiency of the data is influenced. Most seriously, the primers which are easy to form primer dimers seriously affect the amplification efficiency of the target amplification region corresponding to the primers, so that the target sequencing depth is low, and the uniformity of the whole amplification system is finally affected. It can be quite unexplained that primer dimers determine how good the multiplex PCR target capture sequencing is.

Clean Plex dimer elimination technology from Paragon, which employs specific enzymes to eliminate primer dimers in multiplex PCR. This method cannot reduce primer dimer by itself, and is a method of enzymatically digesting after primer dimer is produced. Firstly, the method adopts a mode of removing primer dimers afterwards, the primer dimers generated in the multiplex PCR process can greatly influence the efficiency and the uniformity of PCR amplification, and secondly, the method needs to additionally adopt a digestion step, so the operation is more complicated.

Disclosure of Invention

The invention provides a PCR primer, a PCR amplification method and application, which can greatly reduce primer dimer in the multiple PCR process by inhibiting the primer dimer from being used as a template for subsequent PCR amplification through the self-neck ring structure of the primer dimer.

According to a first aspect, in one embodiment there is provided a primer pair for PCR, the primer pair comprising a forward primer and a reverse primer both comprising a same fixed sequence, and the forward primer and the reverse primer each comprising a specific primer sequence that is different from each other.

In a preferred embodiment, the above-mentioned fixed sequence is located at the 5' end of the specific primer sequence.

In a preferred embodiment, the forward primer and the reverse primer each further comprise an adaptor sequence.

In a preferred embodiment, each of the primers comprises, in order from 5 'to 3': the above linker sequence, the above immobilization sequence and the above specific primer sequence; or the above-mentioned immobilization sequence, the above-mentioned linker sequence and the above-mentioned specific primer sequence.

In a preferred embodiment, the length of the above-mentioned fixed sequence is 15 to 40nt, more preferably 18 to 26 nt.

In a preferred embodiment, the Tm of the above-mentioned fixed sequence is 55 to 65 ℃.

According to a second aspect, there is provided in one embodiment a primer combination for multiplex PCR, the primer combination comprising a plurality of pairs of primers, wherein at least some of the forward and reverse primers of said pairs of primers comprise a same fixed sequence, and wherein the different primers each comprise a specific primer sequence that differs from each other.

In a preferred embodiment, all of the pairs of primers comprise a single fixed sequence.

In a preferred embodiment, the immobilization sequence is located 5' to the specific primer sequence.

In a preferred embodiment, each of the primers further comprises a linker sequence.

In a preferred embodiment, the above-mentioned linker sequence of the forward primers in all the pair primers is the same, and the above-mentioned linker sequence of the reverse primers in all the pair primers is the same.

In a preferred embodiment, the length of the above-mentioned fixed sequence is 15 to 40 nt.

In a preferred embodiment, the length of the fixed sequence is 18 to 26 nt.

In a preferred embodiment, the fixed sequence has a Tm of 60 ℃.

In a preferred embodiment, the primer combination comprises SEQ ID NO: 1-34.

According to a third aspect, there is provided in one embodiment a method of PCR amplification, the method comprising performing PCR amplification using a primer pair of the first aspect or a primer combination of the second aspect.

In a preferred embodiment, the method further comprises further amplifying the PCR amplified product using a universal primer.

According to a fourth aspect, there is provided in one embodiment a primer pair of the first aspect or a primer combination of the second aspect for use in target region capture by PCR amplification.

According to a fifth aspect, an embodiment provides a method of reducing primer dimers in a PCR amplification, the method comprising: performing PCR amplification using the primer pair of the first aspect or the primer combination of the second aspect, such that the generated primer dimers combine with each other to form a neck-loop structure in the immobilized sequences in each primer dimer during subsequent denaturation annealing, thereby preventing subsequent PCR reaction.

According to a sixth aspect, there is provided in one embodiment a target area capture method, the method comprising: performing PCR amplification using the primer pair of the first aspect or the primer combination of the second aspect.

According to a seventh aspect, there is provided in one embodiment a method of library construction, the method comprising: performing PCR amplification using the primer pair of the first aspect or the primer combination of the second aspect.

The primers in the primer pair or the primer combination comprise a section of same fixed sequence, during the PCR process, if dimers are generated between the primers, a complementary sequence exists in a dimer product, the length of the dimer is generally very short, the sequences can be rapidly combined in subsequent denaturation annealing to form a neck ring structure, the subsequent PCR reaction can be prevented, for a normal specific product, although a complementary sequence also exists in the structure, the complementary sequence is long enough, the neck ring structure is difficult to form, and the inhibition of the primer dimer is achieved through the difference of two annealing. Therefore, the primer dimer in the PCR process is greatly reduced by using the primer combination for PCR amplification.

Drawings

FIG. 1 is a schematic diagram of the structure of primer dimer inhibition in an example of the present invention;

FIG. 2 is a schematic representation of primer dimer inhibition in the second generation sequencing library preparation according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of the structure of a sequencing library specific primer in an embodiment of the present invention;

FIG. 4 is a diagram showing the detection result of Agilent 2100 by a conventional method;

FIG. 5 is a diagram showing the detection result of Agilent 2100 in the sequencing library in the embodiment of the invention;

FIG. 6 shows the ratio of the primer dimer filtered out by the method of the present invention and the conventional method.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other materials, methods.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

As shown in FIG. 1, the primer set for ordinary PCR (i.e., PCR performed by one pair of primers) or the forward primer and the reverse primer for multiplex PCR pair primer of the embodiment of the present invention each include a fixed sequence (i.e., the same sequence), in the course of ordinary PCR, or multiplex PCR, if a dimer is generated between primers, the dimer product will have a complementary sequence, since the dimer is generally short, complementary sequences will rapidly bind themselves during subsequent denaturing annealing (i.e., after the second amplification cycle), forming a neck-loop structure that prevents subsequent PCR reactions, whereas for normal specific products, although there is a complementary sequence in the structure, the complementary sequences are sufficiently distant that it is difficult to form a neck loop structure, and inhibition of primer dimer is achieved by the difference in annealing of the two products.

Specifically, as shown in FIG. 2, the forward and reverse primers form primer dimers through primer complementation and amplification in the first cycle of PCR, and the specific primers bind to the target region to amplify normal specific products. In the second cycle of PCR and subsequent cycles, primer dimer denaturation annealing, because two complementary fixed sequences are close to each other, a primer dimer neck ring structure is formed, and further generation of primer dimer is inhibited.

In one embodiment of the present invention, a primer set for PCR is provided, wherein the forward primer and the reverse primer of the primer set comprise a same fixed sequence, and the forward primer and the reverse primer each comprise a specific primer sequence different from each other.

In the embodiment of the present invention, the fixed sequence and the specific primer sequence may be interconnected in a suitable manner to form a primer sequence, for example, they may be directly connected or may be connected by several base sequences between them to form a primer sequence. In a preferred embodiment, the immobilization sequence is located 5' to the specific primer sequence.

In other embodiments, the forward primer and the reverse primer of the primer pair further each comprise an adaptor sequence. Each primer comprises the following components from 5 'end to 3' end in sequence: a linker sequence, an immobilization sequence and a specific primer sequence; or a fixed sequence, a linker sequence and a specific primer sequence. In some embodiments, the length of the fixed sequence in the forward primer and the reverse primer of the primer pair is 15 to 40nt, and the Tm value of the fixed sequence is preferably 55 to 65 ℃.

In one embodiment of the present invention, a primer set for multiplex PCR is provided, the primer set comprising a plurality of pairs of primers, at least some of the primers of the plurality of pairs of primers comprising a same fixed sequence, and different primers each comprising a specific primer sequence different from each other.

The primer combination of the present embodiment comprises a plurality of pairs of primers, each pair of primers comprising a forward primer and a reverse primer for amplifying one target fragment (target region), and different pairs of primers for amplifying different target fragments (target regions), i.e., so-called "multiplex PCR" amplification. The different primers each comprise a specific primer sequence that is different from each other and that is capable of specifically binding to different target fragments (target regions) to achieve specific amplification.

It should be noted that different primer pairs have different tendencies to form primer dimers, some are prone to form primer dimers, and some are not prone to form primer dimers. Thus, at least some of the primers in the plurality of pairs of primers comprise the same fixed sequence, and "partial primers" refer to primers that have formed primer dimers. These "partial primers" may be, for example, 1, 2, 3, 5, 8, 20 or 100 primers. Of course, in a preferred embodiment, the primers of each of the multiple pairs all comprise a segment of the same fixed sequence, such that primer dimers of each pair inhibit further production of primer dimers by forming dimer neck ring structures.

In a preferred embodiment, each primer further comprises a respective linker sequence. The linker sequence may be, for example, a sequencing linker sequence suitable for use in various sequencing platforms. The linker sequences of the forward and reverse primers of each pair of primers may be the same or different, preferably different. The linker sequences of the forward primers may be the same or different, preferably the same, between different primer pairs. Similarly, the linker sequences of the reverse primers may be the same or different, preferably the same, between different primer pairs.

In a more preferred embodiment, as shown in FIG. 3, each primer comprises, in order from the 5 'end to the 3' end: a linker sequence, an immobilization sequence and a specific primer sequence; or a fixed sequence, a linker sequence and a specific primer sequence.

In general, in the present embodiment, the length of the fixed sequence on the primer may not be particularly limited. However, in a preferred embodiment of the present invention, the length of the fixed sequence is 15 to 40nt (bases), and the Tm value is preferably 55 to 65 ℃. In a more preferred embodiment, the length of the fixed sequence is 18 to 26nt, more preferably 18nt, and the Tm value is 60 ℃. In a preferred embodiment, the Tm of the fixed sequence is substantially the same as the Tm of the primer. The GC content is relatively high and the sequence length is as short as possible.

In a preferred embodiment of the invention, a fixed sequence is added to the 5' end of each of one or more pairs of specific primers, the fixed sequence has a length of 15-40nt and a Tm value of 55-65 ℃, and preferably, the fixed sequence has a length of 18nt and a Tm value of 60 ℃. For the method of constructing a sequencing library of interest by multiplex PCR using a two-step method, it is preferable to add an 18nt immobilized sequence, and the first round of the multiplex specific primer structure is shown in FIG. 3.

In a preferred embodiment, a fixed sequence is added to the 5' end of all specific primers in multiplex PCR, and due to the length inconsistency between the target amplicon and the primer dimer, the primer dimer is sufficiently short apart to form a neck ring structure during PCR so that subsequent PCR reaction can not be performed, and further generation of the primer dimer is inhibited.

In one embodiment, the invention provides a method of PCR amplification comprising performing a general PCR, or multiplex PCR amplification using a primer pair or primer combination of the invention.

The PCR amplification method of the present invention may be or may be used for the construction of a target sequencing library, particularly a method for constructing a target sequencing library by multiplex PCR using a two-step method, in which in the first PCR amplification, the specific primer sequence having a fixed sequence of the present invention is used for amplification, and in the second PCR amplification, the universal primer is used for amplification.

In the target sequencing library construction, the fixed sequence and the adaptor sequence on the primer in the primer pair or primer combination of the present invention may be a part of a common or overlapping sequence, for example, a part of the sequence on the sequencing adaptor sequence as the fixed sequence or a part of the fixed sequence.

In one embodiment of the invention there is provided the use of a primer pair or primer combination of the invention for target region capture by PCR amplification. Such use is particularly the use of target region capture library construction.

The primer of the present invention can reduce primer dimer in PCR amplification, and therefore in one embodiment of the present invention, there is provided a method for reducing primer dimer in PCR amplification, comprising: the primer pair or the primer combination is used for PCR amplification, so that the generated primer dimers are combined with each other to form a neck ring structure in the fixed sequence in each primer dimer in the subsequent denaturation annealing process, and the subsequent PCR reaction is prevented.

In one embodiment of the present invention, a target area capturing method is provided, including: PCR amplification is performed using the primer pairs or primer combinations of the invention. In the present invention, the target region may be any nucleic acid sequence region of interest, a typical but non-limiting example being a target gene on genomic DNA from any species. The PCR amplification using the primer pair or primer combination of the present invention results in a captured target region, which may be further amplified in some application scenarios.

In one embodiment, the invention provides a library construction method, comprising: PCR amplification is performed using the primer pairs or primer combinations of the invention. In the present invention, the library may be a nucleic acid library of any suitable use, including but not limited to a sequencing library. The products of the PCR amplification using the primer pairs or primer combinations of the invention can be used as the final library, for example, in the case where the primer combinations of the invention comprise a sequencing linker sequence, the PCR amplification products will carry a sequencing linker sequence, and such amplification products can be subjected to in silico sequencing. However, in some application scenarios, the PCR amplification products may be further subjected to other operations, such as general amplification, circularization, etc., to obtain a sequencing library suitable for on-machine sequencing.

The technical solutions of the present invention are described in detail below by way of examples, and it should be understood that the examples are only illustrative and should not be construed as limiting the scope of the present invention.

Example 1

Designing a primer: 17 pairs of primers were designed for the tumor-associated hot spot region of the ATM gene. For the conventional method, the following primers were designed: adding different sequencing joint sequences to the 5' ends of a pair of specific primers; for the method of the invention, according to the optimized primer design: a fixed sequence is added to the 5' end of a pair of specific primers, and then a different sequencing linker sequence is added to form two tumor-associated gene hot spot mutation detection "panels" (panels), namely, a subsequent specific primer pool.

The experimental process comprises the following steps: the 'panel' is used for preparing libraries of YanhuanggDNA standard products (10-20ng), each time is repeated for 4 times, high-throughput sequencing is carried out after the preparation of the libraries is completed, obtained sequencing data are compared, and primer dimers are mainly compared.

1. First round of PCR, PCR was performed using QIAGEN Multiplex PCR Kit (Cat number/ID: 206143).

A reaction system shown in the following Table 1 was disposed in a 200. mu.L PCR tube:

TABLE 1

Components	Dosage of
Yanhuang gDNA	20μL
2 × PCR reaction enzyme	25μL
Specific primer pool	5μL
Total amount of	50μL

The pool of specific primers used in the method of the present invention was obtained by equimolar mixing of primers 10. mu.M shown in Table 2, wherein underlined sequences represent fixed sequences, sequences preceding fixed sequences represent linker sequences, and sequences following fixed sequences (linker-identical sequences may be a part of fixed sequences) represent specific primer sequences. TABLE 2 primers for the method of the invention

The pool of specific primers used in the conventional method was obtained by equimolar mixing of primers 10. mu.M shown in Table 3, and the sequences included two parts, linker sequence and specific primer sequence, without fixed sequence.

TABLE 3 primers for conventional methods

The first round of PCR amplification was performed according to the procedure of table 4 below:

TABLE 4

mu.L of magnetic beads (Agencour AMPure XP magnetic beads available from Beckman Co., Ltd., product No. A63881) was added to the obtained PCR product and purified, and the obtained product was dissolved in 20. mu.L of TE solution.

For the second round of PCR, PCR was performed using QIAGEN Multiplex PCR Kit (Cat number/ID: 206143).

A reaction system shown in the following Table 5 was arranged in a 200. mu.L PCR tube:

TABLE 5

Components	Dosage of
Reactant of the last step	20μL
2 × PCR reaction enzyme	25μL
Universal primer 1	2.5μL

Universal primer 2	2.5μL
Total amount of	50μL

Wherein, the universal primer 1 and the universal primer 2 are shown in the following table 6:

TABLE 6 Universal primer sequences

Universal primer 1, 5' end phosphorylation for subsequent BGISEQ-500 library circularization.

# Universal primer 2, 10bp N bases as library tag sequences.

A second round of PCR amplification was performed according to the procedure of table 7 below:

TABLE 7

The size of the product was measured by Agilent 2100, and the results are shown in FIGS. 4 and 5, and the solubility was checked by library quality using Qubit 2.0.

2. Sequencing on machine

All the obtained products are standardized and mixed in equal quantity, and the library obtained by mixing is subjected to parallel sequencing, a sequencing platform BGISEQ-500 and a sequencing type PE 100.

3. Data analysis

The analysis step includes the steps of filtering the adaptor primer sequences, and comparing the bases.

As a result: as shown in Table 8 and FIG. 6, the ratio of primer dimers (0.9. + -. 0.5%) obtained by the method of the present invention was significantly smaller than that (49. + -. 1%) obtained by the conventional method. In the conventional method, primer dimer consumes the primer in the system, which leads to the reduction of the amplification efficiency of the target region, thereby affecting the proportion of the amplified region.

TABLE 8

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

Claims

A primer pair for PCR, wherein a forward primer and a reverse primer in the primer pair both comprise a same fixed sequence, and the forward primer and the reverse primer each comprise a specific primer sequence that is different from each other.
The primer pair of claim 1, wherein the immobilization sequence is located 5' to the specific primer sequence.
The primer pair of claim 1, wherein the forward primer and the reverse primer each further comprise an adaptor sequence.
The primer pair of claim 1, wherein each of the primers comprises, in order from 5 'to 3': the adaptor sequence, the immobilization sequence and the specific primer sequence; or the immobilization sequence, the linker sequence and the specific primer sequence.
The primer pair of claim 1, wherein the length of the fixed sequence is 15 to 40 nt.
The primer pair of claim 5, wherein the length of the fixed sequence is 18 to 26 nt.
The primer pair according to claim 1, wherein the fixed sequence has a Tm value of 55 to 65 ℃.
A primer combination for multiplex PCR, wherein said primer combination comprises a plurality of primer pairs, wherein at least some of the forward and reverse primers of said primer pairs comprise a same fixed sequence, and wherein different primers each comprise a specific primer sequence that differs from each other.
The primer combination of claim 8, wherein all of said plurality of primer pairs comprise a same fixed sequence.
The primer combination of claim 8, wherein the immobilization sequence is located 5' to the specific primer sequence.
The primer combination of claim 8, wherein each of said primers further comprises an adaptor sequence.
The primer combination of claim 11, wherein the adaptor sequence of the forward primer of all pair primers is the same and the adaptor sequence of the reverse primer of all pair primers is the same.
The primer combination of claim 12, wherein each of the primers comprises, in order from 5 'to 3': the adaptor sequence, the immobilization sequence and the specific primer sequence; or the immobilization sequence, the linker sequence and the specific primer sequence.
The primer combination of claim 8, wherein the length of the fixed sequence is 15-40 nt.
The primer combination of claim 8, wherein the fixed sequence has a Tm of 55-65 ℃.
The primer combination of claim 8, wherein the primer combination comprises the sequence of SEQ ID NO: 1-34.
A method of PCR amplification comprising PCR amplification using a primer pair according to any one of claims 1 to 7 or a primer combination according to any one of claims 8 to 16.
The method of PCR amplification of claim 17 further comprising further amplifying the PCR amplified product using universal primers.
Use of a primer pair according to any one of claims 1 to 7 or a primer combination according to any one of claims 8 to 16 for target region capture by PCR amplification.
A method of reducing primer dimer in a PCR amplification, the method comprising: performing PCR amplification using the primer pair of any one of claims 1 to 7 or the primer combination of any one of claims 8 to 16, such that the immobilized sequences in each primer dimer bind to each other to form a neck-loop structure during subsequent denaturing annealing to prevent subsequent PCR reactions.
A target area capture method, the method comprising: PCR amplification using the primer pair of any one of claims 1 to 7 or the primer combination of any one of claims 8 to 16.
A method of library construction, the method comprising: PCR amplification using the primer pair of any one of claims 1 to 7 or the primer combination of any one of claims 8 to 16.