阅读说明：本技术 两步pcr技术 (Two-step PCR technique ) 是由 苗鑫垚 沈悦生 龚晓娟 赵梓丞 李梦瑶 贺小兰 于 2021-01-20 设计创作，主要内容包括：本发明公开了一种两步PCR技术,包括如下步骤：第一步,扩增特异性靶点：在此部分混合DNA样品和特异性引物对混合液,使正反向引物的通用序列分别连接到由特异性引物所确定的特异性扩增子的两侧,这一部分反应也被称为第一步PCR；第二步,富集靶点：在富集靶点也就是第二步PCR部分,我们在第一步连接的通用序列后连接了接头。连接好的接头通过第二步PCR进行扩增并纯化,混合到单个试管中并进行纯化。本发明通过更简单的操作步骤造就了其高于市场上通过文库构建测序方法的数据利用率。(The invention discloses a two-step PCR technology, which comprises the following steps: first, amplification of specific targets: mixing the DNA sample and the specific primer pair mixture solution in the part to ensure that the universal sequences of the forward primers and the reverse primers are respectively connected to two sides of the specific amplicon determined by the specific primers, wherein the part of the reaction is also called as first-step PCR; step two, enriching target spots: in the enrichment target, i.e., the second PCR portion, we ligated the linker after the universal sequence ligated in the first step. The ligated adaptors were amplified and purified by a second step of PCR, mixed into a single tube and purified. The invention has the advantage that the data utilization rate is higher than that of a library construction sequencing method in the market through simpler operation steps.)

1. A two-step PCR technique, comprising the steps of:

first, amplification of specific targets: mixing the DNA sample and the specific primer pair mixture solution in the part to ensure that the universal sequences of the forward primers and the reverse primers are respectively connected to two sides of the specific amplicon determined by the specific primers, wherein the part of the reaction is also called as first-step PCR;

step two, enriching target spots: in the enrichment target, i.e., the second PCR part, we ligated the linker after the universal sequence ligated in the first step;

step three, mixing the libraries: the enrichment product is quantified by a fluorescence quantification method, and sample information and concentration information are recorded;

fourth, dilution of the library: the pooled library products were quantified using fluorescent quantitation methods and diluted to 10 ng/. mu.L for pre-sequencing preparation according to the on-machine protocol of the different sequencing platforms.

2. The two-step PCR technique of claim 1, wherein the amount of DNA input for amplification of a specific target is 1-10 ng.

3. A two-step PCR technique according to claim 1, wherein the ligated linkers are amplified and purified by the second PCR step, mixed into a single tube and purified while enriching the target.

4. A two-step PCR technique as claimed in claim 1, wherein the quantified samples are mixed in equal proportions during library mixing.

Technical Field

The invention relates to the field of biology, in particular to a two-step PCR technology.

Background

The polymerase chain reaction PCR is a method for in vitro enzymatic synthesis of specific DNA fragment, and is characterized by that it uses the reactions of high-temp. denaturation, low-temp. annealing (renaturation) and proper-temp. extension to form a period, and makes them implement circulation to make the target DNA be quickly amplified, and has the characteristics of strong specificity, high sensitivity, simple operation and saving time. It can be used for basic research of gene separation, cloning and nucleic acid sequence analysis, and also for diagnosis of diseases or any places with DNA and RNA. PCR is also known as cell-free molecular cloning or in vitro primer directed enzymatic amplification of specific DNA sequences.

The DNA probe (DNA probe) is the most commonly used nucleic acid probe, which is single-stranded or double-stranded DNA with a length of several tens to several hundreds or even thousands of base pairs, and is labeled with a specific tracer (e.g., a isotope, an enzyme, or a chromogen); under proper p-value, temperature and ionic strength, the DNA probe can be combined (hybridized) with complementary non-labeled single-stranded DNA or RNA in a sample to be tested by hydrogen bond to form a double-stranded complex (hybrid) by utilizing the denaturation and renaturation of molecules and the high accuracy of base complementary pairing. The principle of the hybridization capture technology is that according to the DNA sequence complementary principle, a specific probe complementary with a target sequence is designed and synthesized on a solid phase or liquid phase chip, the sample genome DNA is broken, a sequencing joint is added to hybridize with the probe, finally a target region is captured, and then a library is recovered and constructed and is directly sequenced in a high-throughput manner. The solid phase hybridization method, in which a probe is immobilized on a solid support, is typically represented by a gene chip. And after hybridization, eluting the DNA fragment which is not hybridized, eluting the DNA hybridized with the probe, amplifying and constructing a high-throughput sequencing library. The liquid phase hybridization capture technology is understood as that in a solution environment, target DNA is directly hybridized with a probe with a biotin label, a target fragment is anchored on a microbead with avidin through biotin-avidin reaction, the subsequent processes are elution, enrichment, amplification, sequencing library construction and the like, and the current liquid phase hybridization system gradually becomes the mainstream of gene capture.

However, the above target fragment capture methods all require a library construction step to perform the computer sequencing, and the success or failure of the library construction is often the most important step before the sequencing and the most time-consuming step, and is directly related to the success of the sequencing. The aim of constructing the library is to connect the wanted adapters at the two ends of the target fragment, and the library structure needs to meet the on-machine requirements of different kinds of sequencers for sequencing.

Accordingly, the present invention provides a two-step PCR technique to solve the problems set forth in the background art described above.

Disclosure of Invention

The present invention is directed to a two-step PCR technique to solve the problems of the background art.

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

a two-step PCR technique comprising the steps of:

1. the method comprises the following steps:

first, amplification of specific targets: this part of the reaction, in which the DNA sample and the mixture of specific primer pairs are mixed so that the universal sequences of the forward and reverse primers are ligated to both sides of the specific amplicon determined by the specific primers, is also referred to as the first PCR step.

Step two, enriching target spots: in the enrichment target, i.e., the second PCR portion, we ligated the linker after the universal sequence ligated in the first step.

Step three, mixing the libraries: the enrichment product is quantified by a fluorescence quantification method, and the sample information and the concentration information are recorded.

Fourth, dilution of the library: the pooled library products were quantified using fluorescent quantitation methods and diluted to 10 ng/. mu.L for pre-sequencing preparation according to the on-machine protocol of the different sequencing platforms.

As a still further scheme of the invention: the input amount of DNA is 1-10ng when amplifying the specific target.

As a still further scheme of the invention: the ligated adaptors are amplified and purified by a second step of PCR, mixed into a single tube and purified when enriching for the target.

As a still further scheme of the invention: when the library is mixed, the quantified samples are mixed according to equal ratio

Compared with the prior art, the invention has the beneficial effects that:

1. the method is simple and convenient to operate, short in time consumption and suitable for the conditions with special requirements on timeliness: according to the invention, the target fragment amplification and the addition of the sequencing adaptor are completed in two-step PCR by a primer fusion technology, so that the operation steps are simplified, the time consumption is reduced, and the condition with higher requirement on timeliness is met.

2. The invention has the advantage that the data utilization rate is higher than that of a library construction sequencing method in the market through simpler operation steps.

3. The invention has low cost and is suitable for various sequencing platforms: the invention relies on the second-generation sequencing platform and has universality for various sequencing platforms, thereby endowing the advantages of high cost, wide adaptability and the like.

4. The method has lower required DNA amount, can realize accurate typing even when the required DNA amount is as low as 78pg, and needs 50-100ng in traditional library building.

Drawings

FIG. 1 is a technical scheme of a two-step PCR technique.

FIG. 2 is a schematic diagram of the principle of a two-step PCR technique.

FIG. 3 is a graphical representation of data utilization ratios for different approaches in a two-step PCR technique.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example one

In the embodiment of the invention, the two-step PCR technology comprises the following steps:

first, amplification of specific targets: this part of the reaction, in which the DNA sample and the mixture of specific primer pairs are mixed so that the universal sequences of the forward and reverse primers are ligated to both sides of the specific amplicon determined by the specific primers, is also referred to as the first PCR step.

Step two, enriching target spots: in the enrichment target, i.e., the second PCR portion, we ligated the linker after the universal sequence ligated in the first step.

Step three, mixing the libraries: the enrichment product is quantified by a fluorescence quantification method, and the sample information and the concentration information are recorded.

Fourth, dilution of the library: the pooled library products were quantified using fluorescent quantitation methods and diluted to 10 ng/. mu.L for pre-sequencing preparation according to the on-machine protocol of the different sequencing platforms.

Further, the input amount of DNA is 1-10ng when amplifying the specific target spot, the connected joints are amplified and purified by the second step of PCR when enriching the target spot, mixed into a single test tube and purified, and the quantified samples are mixed according to equal ratio when mixing the library

The following table shows the time required before the different methods/kits are loaded:

as shown in the table above, when the 2-step PCR technology is adopted, the total time consumed before the PCR technology is applied is less than that of the construction of a BGISEQ-500 library and is far less than that of the characteristic preparation of a Foren SeqTMDNA.

Example two

FIG. 3 is a graph of data utilization ratios for different methods, where noise represents sequencing noise.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.