CN107385065B

CN107385065B - Method for amplifying nucleic acid and use thereof

Info

Publication number: CN107385065B
Application number: CN201710702484.8A
Authority: CN
Inventors: 郭金海; 常爽爽; 刘进稳; 曾沃坦
Original assignee: Beijing Eagt Research Institute Co ltd
Current assignee: Beijing Eagt Research Institute Co ltd
Priority date: 2017-08-16
Filing date: 2017-08-16
Publication date: 2020-07-28
Anticipated expiration: 2037-08-16
Also published as: CN107385065A

Abstract

The invention discloses a method for amplifying nucleic acid and application thereof. The method for amplifying the nucleic acid comprises the steps of carrying out primer extension on an extension reaction template by using an extension primer to obtain an extension product, wherein the extension product is the amplified nucleic acid; the primer extension is composed of n cyclic extension reactions, wherein n is a natural number from 5 to 25; each cyclic extension reaction of the n cyclic extension reactions consists of two steps, and the first step of each cyclic extension reaction of the n cyclic extension reactions is carried out under the condition that the reaction temperature is 94 ℃ and the reaction time is 30 seconds; the reaction temperature and the reaction time of the second step of the n-cycle extension reaction are increased, the rate of increase of the reaction temperature of the second step is 0.5 ℃/cycle, and the rate of increase of the reaction time of the second step is 20 to 30 seconds/cycle. The method for amplifying nucleic acid of the present invention can improve the amplification efficiency of a nucleic acid extension reaction template.

Description

Method for amplifying nucleic acid and use thereof

Technical Field

The invention relates to a method for amplifying nucleic acid in the field of biotechnology and application thereof.

Background

EGFR is the epidermal growth factor receptor, and EGFR mutations occur in 57% of patients with glioblastoma. Moreover, EGFR mutations are of various types and involve various mutation patterns such as gene amplification, gene rearrangement, indels, point mutations, and the like. Is common in glioblastoma as epidermal growth factor receptor type iii (EGFRv iii) mutations, with about 50% of EGFR amplifying mutations accompanied by EGFRv iii mutations. The current major detection methods for EGFRv type iii mutations are 1) fluorescent quantitative PCR detection methods at the RNA level; 2) and (3) detecting the somatic mutation by a second-generation sequencing method. The fluorescent quantitative PCR detection method at RNA level cannot identify mutations at genomic DNA level, and RNA is easily degraded with respect to DNA, and has a detection sensitivity for RNA far lower than that for DNA. And the cost for detecting the somatic mutation by the second-generation sequencing method is relatively high. Therefore, the detection of EGFRv III mutation at the DNA level has important significance and clinical diagnosis value.

EGFRvIII mutations involve sequence rearrangements of the EGFR gene, which are expressed at the mRNA level as deletions of exons 2-7, but are highly variable at the DNA level and are very complex, with an upstream breakpoint within a first intron (the intron is about 123kb long), and a downstream breakpoint within a seventh intron, whose first intron has a large span, resulting in strong randomness of the positions of the EGFRvIII mutation breakpoints, and therefore the difficulty of detecting EGFRvIII mutations at the DNA level is great, and there are few kits for detecting EGFRvIII mutations at the current market, but it is not entirely clear from the disclosure of L oriderFrek that EGFRvIII mutations occur mainly between Alu repeats, an Alu repeat sequence in intron 7, all breakpoints downstream of EGFRvIII mutations occur near this sequence, whereas upstream breakpoints are highly related to the first one, and upstream primers containing more than 1 ng repeats in the first intron, and PCR with a specific primer set of primers 3580% upstream primers are designed to react with each other, and the upstream primers are not specifically designed to react with upstream primers of the upstream and downstream primers of the upstream primers, and downstream primers, and the primers are not specifically designed by PCR method, and the PCR method for detecting AlFreuk PCR, which the upstream primers are designed by a specific primer mix PCR method, and the upstream primer mix the upstream primer set of primers, and the PCR method, the primers, and the primers, and the primers, the primers are not only for detecting AlFreup primers, and the primers are not used for detecting Alru primers for PCR method.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the amplification efficiency of the nucleic acid extension reaction template.

In order to solve the above-mentioned problems, the present invention provides a method for amplifying a nucleic acid, which is also called a time-temperature-increasing cyclic extension reaction method.

The method for amplifying nucleic acid provided by the invention comprises the steps of carrying out primer extension on an extension reaction template by using an extension primer to obtain an extension product, wherein the extension product is amplified nucleic acid; the primer extension is composed of n cyclic extension reactions, wherein n is a natural number from 5 to 25; each cyclic extension reaction of the n cyclic extension reactions consists of two steps, and the first step of each cyclic extension reaction of the n cyclic extension reactions is carried out under the conditions that the reaction temperature is 90-98 ℃ and the reaction time is 15-40 seconds; the reaction temperature and the reaction time of the second step of the n cycle extension reactions are increased, the rate of increase of the reaction temperature of the second step is 0.2 ℃ to 2 ℃/cycle, and the rate of increase of the reaction time of the second step is 10 to 120 seconds per cycle; the reaction temperature of the second step of the first cycle extension reaction of the n cycle extension reactions is T, the reaction time is 1 minute, the T is greater than or equal to the Tm value of the extension primer, and the T is between 68 ℃ and 75 ℃.

In the method for amplifying a nucleic acid, the primer extension (primer extension) is a process of synthesizing a strand complementary to a template (RNA or DNA) from the 3 ' -OH end of a primer (extension primer) bound to the extension reaction template by connecting nucleotides one by one in the base pairing principle by the action of a nucleic acid polymerase from the 5 ' → 3 ' direction.

In the above method for amplifying a nucleic acid, the extension primer is a small piece of single-stranded DNA, and serves as a polynucleotide chain which serves as a starting point for DNA replication and as a starting point for extension of each polynucleotide chain during a nucleic acid synthesis reaction.

In the method for amplifying a nucleic acid, the extension reaction template is a nucleic acid (DNA or RNA) containing a desired extension fragment.

In the method for amplifying a nucleic acid as described above, the reaction temperature of the first step of each cycle of the extension reaction may be 92 ℃ to 96 ℃, 93 ℃ to 95 ℃, or 94 ℃; the reaction time of the first step of the extension reaction may be 20 to 35 seconds, 25 to 35 seconds, or 30 seconds per cycle.

In the method for amplifying a nucleic acid, the reaction temperature of the second step may be increased at a rate of 0.3 ℃/min-1.5 ℃/min, 0.4 ℃/min-1.0 ℃/min, 0.5 ℃/min-0.8 ℃/min, or 0.5 ℃/min, and the reaction time of the second step may be increased at a rate of 20 sec/min-100 sec/min, 30 sec/min-80 sec/min, 30 sec/min-50 sec/min, 30 sec/min-40 sec/min, 20 sec/min-30 sec/min, or 30 sec/min.

In the method for amplifying a nucleic acid, n may be a natural number of 10 to 25, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

In the method for amplifying nucleic acid, the reaction temperature in the second step of the first cyclic extension reaction of the n cyclic extension reactions is T ℃ and the reaction time is 60 seconds, the reaction temperature in the second step of the second cyclic extension reaction of the n cyclic extension reactions is T +0.5 ℃ and the reaction time is 90 seconds, the reaction temperature in the second step of the third cyclic extension reaction of the n cyclic extension reactions is T +1.0 ℃ and the reaction time is 120 seconds, and … …, the reaction temperature in the second step of the nth cyclic extension reaction of the n cyclic extension reactions is T +0.5 × (n-1) ° C and the reaction time is 60 seconds +30 × (n-1) seconds.

In the method for amplifying a nucleic acid, the extension reaction template may be DNA (which may be single-stranded or double-stranded) or RNA.

In the method for amplifying a nucleic acid, the extension primer is a single-stranded DNA. The extension primer is one.

The method for amplifying a nucleic acid is a method for amplifying a nucleic acid in vitro.

In the method for amplifying a nucleic acid, the extension reaction template may be derived from any species, such as plants, microorganisms, or animals. The animal can be a mammal, such as a human. When the extension reaction template is derived from an animal, the extension reaction template is an ex vivo animal tissue and/or organ.

The invention also provides a method for preparing the PCR template by using the method for amplifying the nucleic acid.

The method for preparing the PCR template comprises the step of carrying out primer extension on the extension reaction template by using the extension primer according to the method for amplifying the nucleic acid to obtain an extension product, wherein the extension product is the PCR template.

In the above method for preparing a PCR template, the PCR template is a nucleic acid (DNA or RNA) containing a desired PCR amplification fragment.

In the above method for preparing a PCR template, the extension reaction template may be derived from any species, such as plants, microorganisms, or animals. The animal can be a mammal, such as a human. When the extension reaction template is derived from an animal, the extension reaction template is an ex vivo animal tissue and/or organ.

The invention also provides a method for performing polymerase chain reaction by using the method for amplifying nucleic acid.

The polymerase chain reaction method provided by the invention comprises the following steps:

a1) preparing a PCR template according to the method for preparing the PCR template;

a2) and carrying out polymerase chain reaction on the PCR template by using the PCR primer.

In the above polymerase chain reaction method, the polymerase chain reaction may be long-Range PCR (long-Range PCR) or non-long-Range PCR.

In the above polymerase chain reaction method, the long-distance PCR is a PCR capable of amplifying a DNA fragment of 4kb to 10kb and/or a DNA fragment of less than 4 kb. The non-long-range PCR is a PCR that can amplify only a DNA fragment of less than 4 kb.

In the above polymerase chain reaction method, the extension reaction template may be derived from any species, such as plants, microorganisms, or animals. The animal can be a mammal, such as a human. When the extension reaction template is derived from an animal, the extension reaction template is an ex vivo animal tissue and/or organ.

The invention also provides a method for identifying the III type mutant gene of the epidermal growth factor receptor by using the method for amplifying the nucleic acid.

The method for identifying the III type mutant gene of the epidermal growth factor receptor comprises the following steps:

b1) by adopting the method for amplifying the nucleic acid, the genome DNA of the isolated tissue and/or organ of the person to be detected is used as an extension reaction template, and the extension primer is used for carrying out primer extension on the extension reaction template to obtain an extension product;

b2) and (3) taking the extension product as a PCR template, carrying out polymerase chain reaction on the PCR template by using a PCR primer, detecting a specific PCR amplification product, and identifying whether the person to be detected contains the epidermal growth factor receptor III type mutant gene or not according to the sequence of the specific PCR amplification product.

In the method for identifying the epidermal growth factor receptor III type mutant gene, the extension primer is single-stranded DNA with a nucleotide sequence of a sequence 13 in a sequence table; and/or the presence of a gas in the gas,

n is 15; and/or the presence of a gas in the gas,

the reaction temperature of the second step of the first cyclic extension reaction of the n cyclic extension reactions is 72 ℃; and/or the presence of a gas in the gas,

the PCR primer consists of an upstream primer and a downstream primer, and the downstream primer is a single-stranded DNA with a nucleotide sequence of sequence 12 in the sequence table; the upstream primer is a mixture consisting of the following F1-F11:

f1, single-stranded DNA with the nucleotide sequence of sequence 1 in the sequence table;

f2, a single-stranded DNA with a nucleotide sequence of sequence 2 in the sequence table;

f3, a single-stranded DNA with a nucleotide sequence of sequence 3 in the sequence table;

f4, single-stranded DNA with the nucleotide sequence of sequence 4 in the sequence table;

f5, single-stranded DNA with the nucleotide sequence of sequence 5 in the sequence table;

f6, a single-stranded DNA with a nucleotide sequence of sequence 6 in the sequence table;

f7, a single-stranded DNA with a nucleotide sequence of sequence 7 in the sequence table;

f8, a single-stranded DNA with a nucleotide sequence of sequence 8 in the sequence table;

f9, single-stranded DNA with the nucleotide sequence of sequence 9 in the sequence table;

f10, a single-stranded DNA with a nucleotide sequence of sequence 10 in the sequence table;

f11 and the nucleotide sequence is single-stranded DNA of sequence 11 in the sequence table.

In the method for identifying the III-type mutant gene of the epidermal growth factor receptor, the amounts of substances F1-F11 in the upstream primer are the same. In the PCR primer, the mass ratio of the F1 to the downstream primer is 1: 4.

In the above method for identifying the EGFR type III mutant gene, the tissue and/or organ may be a normal tissue and/or organ or may be an abnormal tissue and/or organ. The tissue and/or organ may be blood (e.g., peripheral blood) and/or tumor tissue. The subject may be a mammal, such as a human.

The invention also provides a product for identifying the III type mutant gene of the epidermal growth factor receptor by using the method for amplifying the nucleic acid.

The product for identifying the III-type mutant gene of the epidermal growth factor receptor provided by the invention is K1, K2, K3 or K4:

k1, kit for identifying epidermal growth factor receptor type III mutant genes, the kit contains the extension primer and the PCR primer;

k2, a kit for identifying epidermal growth factor receptor type III mutant genes, wherein the kit contains the PCR primer;

k3, a reagent for identifying epidermal growth factor receptor type III mutant genes, the reagent contains the extension primer and the PCR primer;

k4, and a reagent for identifying epidermal growth factor receptor type III mutant genes, wherein the reagent contains the PCR primer.

In the above products for identifying an epidermal growth factor receptor type iii mutant gene, the kit for identifying an epidermal growth factor receptor type iii mutant gene described in K1 and the reagent for identifying an epidermal growth factor receptor type iii mutant gene described in K3 may contain only the extension primer and the PCR primer, and may further contain other reagents for performing an extension reaction and/or a PCR reaction. The kit for identifying the epidermal growth factor receptor type III mutant gene described in K2 and the reagent for identifying the epidermal growth factor receptor type III mutant gene described in K4 may only contain the PCR primer, and may further contain other reagents for performing PCR reaction.

Any of the following applications of P1 to P3 also fall within the scope of the present invention:

p1, use of the method for amplifying a nucleic acid as described above for performing a polymerase chain reaction;

p2, use of the method for amplifying a nucleic acid as described above for detecting a single nucleotide polymorphism;

p3 and the application of the method for amplifying nucleic acid in gene mutation detection.

In any of the above methods for amplifying a nucleic acid, methods for preparing a PCR template, polymerase chain reaction methods, methods for identifying epidermal growth factor receptor type III mutant genes, and applications of any of P1 to P3, when the extended reaction template thereof is derived from an animal, the purpose thereof may be for disease diagnosis, disease prognosis, and/or disease treatment, and the purpose thereof may also be for non-disease diagnosis, non-disease prognosis, and non-disease treatment; their direct purpose may be to obtain information on the outcome of a disease diagnosis, prognosis of a disease and/or intermediate outcome of a disease treatment, and their direct purpose may be non-disease diagnosis, non-disease prognosis and/or non-disease treatment.

In order to reduce the phenomenon of non-specific amplification, the traditional method is to use a nested PCR method to amplify a target template in a first round, and then use the product of the first round of amplification to amplify in a second round, wherein the two rounds of amplification use different PCR primers, and the primer of the first round of amplification is outside the primer of the second round of amplification. However, since a single upstream primer cannot be designed due to the randomness of the upstream sequence of EGFRv III genomic DNA, the nested PCR method cannot be used, and the present inventors used a one-way extension reaction amplification method for EGFRv III amplification. However, since the efficiency of template amplification by the conventional extension reaction amplification method is low, the present invention provides a method for amplifying nucleic acid, which can greatly improve the efficiency of template amplification by the extension reaction, unlike the conventional extension reaction, in which the extension time and temperature of each cycle of the extension reaction are sequentially increased, and the method for amplifying nucleic acid of the present invention is also called as a time-temperature increasing cycle extension reaction method. The time temperature increment cyclic extension reaction method is particularly suitable for preparing the following templates of PCR reaction: 1. the target PCR amplification fragment is relatively long and contains a plurality of mutation sites; 2. the target PCR amplified fragment contains a plurality of mutation sites although the fragment is not long; 3. the target PCR amplified fragment is longer, although only contains one mutation site, the position of the mutation site is uncertain, and a plurality of possible positions exist; 4. the content of the genomic DNA carrying the target PCR amplification fragment in the tissues and/or organs to be detected is low.

The time-temperature increasing cyclic extension reaction method can improve the amplification efficiency of a nucleic acid extension reaction template, the time-temperature increasing cyclic extension reaction method (scheme 4) can amplify the extension reaction template to 32.86 times, the one-step extension method (scheme 2) and the simple cyclic method (scheme 3) can only amplify the extension reaction template to 3.42 times and 12.32 times respectively (table 4), the time-temperature increasing cyclic extension reaction method is adopted to amplify the extension reaction template, the extension reaction product obtained by amplification is used as a PCR template, the PCR reaction is carried out by adopting the PCR primer, the detection accuracy of the EGFRvIII can be greatly improved, the omission rate (false negative rate) is reduced, the detection accuracy of the FresFresng-Range PCR is carried out by adopting the PCR primer mixture A of L ori and the downstream primer of L ori to carry out Fresng-Range PCR, the detection accuracy of the RvRvRvIII is 15.6%, and the detection accuracy of the RvIrerk mixture B of L ori and the Rerk is 35.7.7%.

Drawings

FIG. 1 is a graph comparing the time-temperature increasing cyclic extension reaction method of the present invention with the one-step extension reaction method and the cyclic extension reaction method.

FIG. 2 shows the results of sequencing the EGFRv III genomic DNA in EGFRv III positive samples.

FIG. 3 shows the results of electrophoretic detection of PCR products of long-Range PCR reaction using the extension products of four extension reaction schemes as PCR templates. In FIG. 3, the non-extension reaction indicates that the extension product of scheme 1 is used as a PCR template, the one-step extension method indicates that the extension product of scheme 2 is used as a PCR template, the cycle extension method indicates that the extension product of scheme 3 is used as a PCR template, and the time-temperature-increment cycle extension method indicates that the extension product of scheme 4 is used as a PCR template.

FIG. 4 shows the results of the electrophoresis of the PCR products of 13 EGFRv III positive samples by the long-Range PCR of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

A typical extension reaction employs a 30-minute extension protocol at 72 ℃ (the one-step extension reaction method in FIG. 1), which is suitable for reverse transcription of RNA, and thus the extension conditions are only one cycle of extension of the target template, which is equivalent to only one template replication process. The extension reaction can also be performed by cyclic extension, which generally has a fixed extension time (e.g., a long extension time, such as 5 minutes, is required for amplifying a long fragment), a fixed extension temperature (72 ℃), and after multiple cycles, the extension product is too long and difficult to melt, thereby causing difficulty in competitive binding of the extension primer and the template, and reducing the efficiency (the cyclic extension reaction method in fig. 1). The time-temperature increasing cyclic extension reaction method specifically comprises the following steps: the first round of extension time was reduced to 1 minute, and as the number of cycles increased, the extension time increased by 30 seconds per each additional cycle, and the temperature increased by 0.5 deg.C (time-temperature increment cyclic extension method in FIG. 1). The advantages of this are: the extension products in the first few rounds are short, easy to melt, and relatively small in competitive binding effect of the primer and the template, and even if the extension products are in competitive binding, the extension products can be used as the primers to continue the reaction and extend the target template sequence, so that the time-temperature increasing cycle extension method is superior to the one-step extension reaction method and the cycle extension reaction method in the amplification efficiency of the template.

The time temperature increment cycle extension reaction method of the present invention is illustrated below by way of example of a method for identifying epidermal growth factor receptor type III mutant genes.

The extension primers and PCR primers in the following examples are shown in Table 1, VIII-F1 to VIII-F11 are the upstream primers of long-Range PCR reaction, and VIII-R1 is the downstream primer of long-Range PCR reaction. VIII-R2 is an extension primer of the time temperature increment cycle extension reaction method. The position where the extension primer VIII-R2 was designed is located downstream of the downstream primer of the long-Range PCR.

TABLE 1 extension primers and PCR primers

Note: the primer sequence is 5 '→ 3' oriented from left to right.

In the following examples

Taq DNA Polymerase was purchased from NEB (cat. No.: M0323L) and used for the long-Range PCR amplification reaction.

The long-Range PCR of the present invention in the following examples was carried out by a method described in an article of L ori Frederick (Analysis of genomic rearrangements associated with EGRFvIII expression requirements in volvement of Alu repeat elements L ori Frederick, et al NeuroOncol.2000 Jul; 2(3):159-63) with minor modifications, in particular, a 50 μ L long-Range PCR Reaction system consisting of 10ng of PCR template, 10. mu.M of 11 upstream primers VIII-F1 to F11, 0.15 μ M of each final concentration, 0.6 μ M of VIII-R1, 10. mu.l of 5X L ong Amp Taq Reaction Buffer, 10. mu.l of 10mM dNs, 1.5. mu.l of TPs, L, Taq 1. mu.l of Taq Polymerase, and no complement of 50 μ l Taq DNA.

The reaction procedure of the Long-Range PCR of the present invention is shown in Table 2:

TABLE 2 reaction procedure for Long-Range PCR of the present invention

Example 1 amplification of nucleic acids Using the time temperature-increasing Cyclic extension reaction method of the present invention

1. Preparation of EGFRv III Positive samples

The method comprises the following steps of (1) taking human glioblastoma tissue, extracting total RNA of the human glioblastoma tissue, identifying EGFRv III gene by using an RT-PCR method (Molecular determination of the genes of Rhabdormas to EGFR kinase inhibitors. N Engl J Med 2005; 353:2012-24.Mellinghoff IK, Wang MY, Vivanco I, et al.), taking human glioblastoma tissue, extracting genomic DNA of the human glioblastoma tissue, and using an L ori Frederick method (Analysis of genetic rearariangement assortment associated with amplified gene of alu repeat genes. L ori degerifick, et al. neuro col.2000Jul; 2(3) EGFRev amplification of EGFRv EGFreuv I. envelope 159-63), sequencing by using primers, verifying that the amplification of the target gene of Rhabdorms III gene is shown in a PCR amplification band, and comparing the result of the content of the Rhabdormoviral gene III gene with the gene of the wild Rhabdormoviral gene III gene of the human glioma III sample (2: 7. the Rfoveol-gene III gene), and the result of the Rbvere-PCR detection of the Rfoveol gene containing the RfvIII gene sequence of the RfvIII gene III sequence, wherein the RfvIII sequence is shown in the Rfve III sample, the Rfvi-III gene sequence of the Rfvi-III sample, the sequence is shown in the Rfve, and the sequence of the Rfvi III gene of the RfvIII sample, and the sequence of the Rfve, and the sequence of the gene of the Rfve III sample is shown in the gene of the RfvIII sample, and the sequence of the Rfve III sample, and the sequence of the wild Rhabdormic sample, and the sequence of the Rfve.III sample, and the sequence of the gene of the sequence of the Rfve.III, and the sequence of the Rfve:

VIII-5-F:5’-GTAGCCTCTGACCCCTAAGGA-3’，

VIII-5-R:5’-AGATGGAGTCTTGCTCTGTCAC-3’，

VIII-5-PROBE:5’-(FAM)-CCTGTAATCCAGGTAATCACAGC-(MGB)-3’。

quantstudio using life corporation^TM3D digital PCR system, specific experimental method refers to QuantStudio^T3D digital PCR system operating instructions.

Diluting human peripheral blood genome DNA only containing EGFR wild type gene with sterile water to 50 ng/mul of genome DNA content, and taking the obtained solution as WT sample; the EGFRvIII positive sample with the EGFRvIII mutation frequency of 34 percent is diluted by sterile water to the genomic DNA content of 50 ng/. mu.l, the obtained solution is the 34 percent EGFRvIII sample, and then the weight ratio of the sample to the total weight of the sample is as follows: the 34% EGFRv iii samples were run according to 12: 5, obtaining a solution which is an EGFRvIII positive sample (for short, 10% EGFRvIII sample) with the EGFRvIII mutation frequency of 10%, then mixing the WT sample and the 10% EGFRvIII sample according to the volume ratio of 9:1 to obtain a solution which is a 1% EGFRvIII positive sample (for short, 1% EGFRvIII sample), then mixing the WT sample and the 1% EGFRvIII sample according to the volume ratio of 9:1 to obtain a solution which is a 0.1% EGFRvIII positive sample (for short, 0.1% EGFRvIII sample).

2. Influence of different extension schemes on the amplification degree of extension reaction template and on the detection sensitivity of EGFRvIII gene

Respectively taking the WT sample, the 0.1% EGFRvIII sample, the 1% EGFRvIII sample and the 10% EGFRvIII sample in the step 1 as extension reaction templates, and respectively carrying out primer extension on the extension reaction templates on a PCR instrument by using extension primers VIII-R2 according to the extension reaction schemes of the following schemes 1 to 4 to obtain extension products, wherein the extension products are amplified nucleic acids.

The extension reaction system of the four extension reaction schemes is 50 mul reaction system, the composition of the 50 mul reaction system is 10ng extension reaction template, the final concentration of extension primer VIII-R2 is 0.2 mul, 10 mul of 5X L ong Amp Taq reaction buffer, 1 mul of 10mM dNTPs, 1 mul of L ong Amp Taq DNA Polymerase, and the nuclease-free water is supplemented to 50 mul.

The reaction procedure for the four extension reaction schemes is as follows:

scheme 1: the extension reaction system does not carry out extension reaction.

Scheme 2: one-step extension method: carrying out the following extension reaction on the extension reaction system: denaturation at 94 ℃ for 5 min, extension at 72 ℃ for 60 min and storage at 4 ℃ last.

Scheme 3: a cyclic extension method: carrying out the following extension reaction on the extension reaction system: firstly, denaturation is carried out for 5 minutes at 94 ℃; the following 15 cycles were then performed: denaturation at 94 ℃ for 30 seconds and extension at 72 ℃ for 5 minutes; then extending for 10 minutes at 72 ℃; finally, the mixture is stored at 4 ℃.

Scheme 4: the time-temperature increasing cyclic extension reaction method of the invention has 15 cyclic extension reactions, and the first step of each cyclic extension reaction is reaction at 94 ℃ for 30 seconds; the reaction temperature and reaction time for the second step were both increased at a rate of 0.5 deg.C/cycle and at a rate of 30 seconds/cycle.

The time-temperature increasing cyclic extension reaction method specifically comprises the following steps: 1) denaturation was first carried out for 5 min at 94 ℃.

2) The following 15 cycles were then performed: cycle 1 is denaturation at 94 ℃ for 30 seconds followed by extension at 72 ℃ for 60 seconds; cycle 2 is denaturation at 94 ℃ for 30 seconds followed by extension at 72.5 ℃ for 90 seconds; cycle 3 is denaturation at 94 ℃ for 30 seconds followed by extension at 73 ℃ for 120 seconds; the 4 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 73.5 ℃ for 150 seconds; the 5 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 74 ℃ for 180 seconds; cycle 6 is denaturation at 94 ℃ for 30 seconds followed by extension at 74.5 ℃ for 210 seconds; the 7 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 75 ℃ for 240 seconds; the 8 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 75.5 ℃ for 270 seconds; cycle 9 is denaturation at 94 ℃ for 30 seconds followed by extension at 76 ℃ for 300 seconds; the 10 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 76.5 ℃ for 330 seconds; cycle 11 is denaturation at 94 ℃ for 30 seconds followed by extension at 77 ℃ for 360 seconds; the 12 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 77.5 ℃ for 390 seconds; the 13 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 78 ℃ for 420 seconds; the 14 th cycle is denaturation at 94 ℃ for 30 seconds followed by extension at 78.5 ℃ for 450 seconds; the 15 th cycle is a denaturation at 94 ℃ for 30 seconds followed by an extension at 79 ℃ for 480 seconds.

3) Extension was carried out at 72 ℃ for 10 minutes.

4) Finally, the mixture is stored at 4 ℃.

TABLE 3 reaction procedure for four extension reaction schemes

The extension product was purified using a DNA purification kit (Tiangen Biochemical Co., Ltd.; Cat. No.: DP214-03), eluted with 50. mu.l of an eluent, and subjected to the digital PCR experiment of step 1 to examine the influence of the four extension reaction protocols on the amplification degree of the extension reaction template.

The purified extension product is used as a PCR template to carry out the long-Range PCR amplification of the invention, and the influence of the four extension reaction schemes on the detection sensitivity of the EGFRv III gene is detected.

The results of the digital PCR experiment of step 1 performed on the extension products of the four extension reaction schemes of the 10% EGFRv iii sample showed that the time-temperature increasing cycle extension reaction method (scheme 4) of the present invention can amplify the extension reaction template 32.86 times, and the one-step extension method (scheme 2) and the simple cycle method (scheme 3) can amplify the extension reaction template only 3.42 times and 12.32 times, respectively (table 4).

TABLE 4 copy number of EGFRvIII Gene of extension products and amplification factor of extension reaction templates for four extension reaction protocols

	Copy number of EGFRv III Gene	Amplification factor of extension reaction template
			Scheme
1	218 copies/. mu.l	1
			Scheme 2	745 copies/. mu.l	3.42
Scheme 3	2686 copies/. mu.l	12.32
			Scheme 4	7163 copies/. mu.l	32.86

The results of the long-Range PCR amplification reaction according to the present invention performed on the extension products of the four extension reaction schemes of the WT sample, the 0.1% egfrviii sample, the 1% egfrviii sample, and the 10% egfrviii sample indicate that the specific PCR product can be obtained in the 0.1% egfrviii sample by using the extension product of the time-temperature increment cyclic extension reaction method (scheme 4) of the present invention as the PCR template, and the specific PCR product cannot be obtained in the 0.1% egfrviii sample by using the extension products of the other three extension reaction schemes as the PCR templates. The time-temperature increasing cyclic extension reaction method can detect low-frequency mutation as low as 0.1%, but the other methods cannot detect the low-frequency mutation, and the time-temperature increasing cyclic extension reaction method can greatly improve the sensitivity (figure 3).

3. Comparison of the PCR primers VIII-F1 to VIII-F11 and VIII-R1 of the present invention with the primer combination of L ori Frederick

13 EGFRv III positive samples (13 genomic DNAs of human glioblastoma tissue containing the EGFRv III gene (exon 2 to 7 deletions compared to the EGFR wild-type gene)) were prepared from 36 human glioblastoma tissues according to the method of step 1, and extension reaction was performed according to the time-temperature increasing cycle extension reaction method of the present invention of step 2 to obtain 13 EGFRv III positive samples as extension products.

The PCR system was performed by using the extension products of the EGFRv III positive samples of 13 cases as PCR templates, using VIII-F1 to VIII-F11 and VIII-R1 in Table 1 as PCR primers of the present invention, performing the long-Range PCR of the present invention, using the extension products of the EGFRv III positive samples of 13 cases as PCR templates, using primer mixture A (primer mixture A) of L ori Frederick and primer mixture B (primer mixture B) (Table 6) (amplification of amplification mutation with amplification deletion of amplification mutation, et al. neuro col.2000 Jul; 2(3) of amplification mutation, 159-63 as primers, using the above-mentioned primer system as upstream primer pair PCR system, using the above-F2 to downstream primer pair PCR system as PCR primers of amplification mutation A-9, using the above-PCR system as upstream primer pair PCR system, using primer pair PCR system with primer pair A-9-PCR system as upstream primer pair PCR system, using primer pair PCR system with primer pair A-PCR system of primer pair A-III primer pair PCR primers of primer pair PCR Reaction, PCR system.

The reaction procedure for the A versus Long-Range PCR and the B versus Long-Range PCR was the same as that for the Long-Range PCR of the present invention, as shown in Table 2.

TABLE 5 primer mixture A

Note: the primer sequence is 5 '→ 3' oriented from left to right.

TABLE 6 primer mixture B

Note: the primer sequence is 5 '→ 3' oriented from left to right.

Electrophoresis detection results show that 11 EGFRvIII positive samples obtain specific PCR products by adopting the long-Range PCR, the accuracy of 11 EGFRvIII positive samples is 84.6%, 2 EGFRvIII positive samples obtain specific PCR products by adopting an A pair specific long-Range PCR, and 11 EGFRvIII positive samples do not obtain specific PCR products by adopting the A pair specific long-Range PCR, so that 2 EGFRvIII positive samples can be detected by adopting the A pair specific long-Range PCR, the accuracy of 13 EGFRvIII positive samples is 15.4%, 13 EGFRvIII positive samples can be detected by adopting a B pair specific long-Range PCR, the accuracy of 1 EGFRvIII positive samples is 1, the accuracy of 12 EGFRvIII positive samples is not obtained, and the accuracy of 13 EGFRvIII positive samples can be greatly improved by adopting a B pair specific long-Range PCR, and the accuracy of 3% fake-Range PCR is 7.7%.

The electrophoresis detection results of 13 cases of EGFRvIII positive samples by the long-Range PCR of the invention are shown in FIG. 4, and 11 cases of EGFRvIII positive samples with the numbers of 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 10#, 11# and 13# obtain specific PCR products, wherein the sizes of the specific PCR products are 3319bp, 4487bp, 968bp, 511bp, 2274bp, 1398bp, 1215bp, 1687bp, 1405bp, 1872bp and 3286bp respectively.

<110> Beijing east Asia-American Gene science and technology institute Co., Ltd

<120> method for amplifying nucleic acid and use thereof

<160>13

<170>PatentIn version 3.5

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Claims

1. A method for amplifying a nucleic acid, comprising: the method comprises the steps of carrying out primer extension on an extension reaction template by using an extension primer to obtain an extension product, wherein the extension product is amplified nucleic acid; the primer extension is composed of n cyclic extension reactions, wherein n is a natural number from 5 to 25; each cyclic extension reaction of the n cyclic extension reactions consists of two steps, and the first step of each cyclic extension reaction of the n cyclic extension reactions is carried out under the conditions that the reaction temperature is 90-98 ℃ and the reaction time is 15-40 seconds; the reaction temperature and the reaction time of the second step of the n cycle extension reactions are increased, the rate of increase of the reaction temperature of the second step is 0.2 ℃ to 2 ℃/cycle, and the rate of increase of the reaction time of the second step is 10 to 120 seconds per cycle; the reaction temperature of the second step of the first cycle extension reaction of the n cycle extension reactions is T, the reaction time is 1 minute, the T is greater than or equal to the Tm value of the extension primer, and the T is between 68 ℃ and 75 ℃.

2. The method of claim 1, wherein: the reaction temperature of the first step of each cyclic extension reaction is 92-96 ℃; the reaction time of the first step of the extension reaction per cycle is 20 to 35 seconds.

3. The method of claim 2, wherein: the reaction temperature of the first step of each cyclic extension reaction is 93-95 ℃; the reaction time of the first step of the extension reaction per cycle is 25 to 35 seconds.

4. The method of claim 3, wherein: the reaction temperature of the first step of each cyclic extension reaction was 94 ℃; the reaction time of the first step of the extension reaction was 30 seconds per cycle.

5. The method according to any one of claims 1-4, wherein: the reaction temperature of the second step is increased at a rate of 0.3 ℃/cycle to 1.5 ℃/cycle, and the reaction time of the second step is increased at a rate of 20 seconds/cycle to 100 seconds/cycle.

6. The method of claim 5, wherein: the reaction temperature of the second step is increased at a rate of 0.4 ℃/cycle to 1.0 ℃/cycle, and the reaction time of the second step is increased at a rate of 30 seconds/cycle to 80 seconds/cycle.

7. The method of claim 6, wherein: the reaction temperature of the second step is increased at a rate of 0.5 ℃/cycle to 0.8 ℃/cycle, and the reaction time of the second step is increased at a rate of 30 seconds/cycle to 50 seconds/cycle.

8. The method of claim 7, wherein: the reaction temperature of the second step was increased at a rate of 0.5 ℃ per cycle, and the reaction time of the second step was increased at a rate of 30 seconds per cycle.

9. The method according to any one of claims 1-4, wherein: the extension reaction template is DNA or RNA.

10. A method of preparing a PCR template comprising primer-extending an extension reaction template with an extension primer according to the method of any one of claims 1 to 9 to obtain an extension product, the extension product being the PCR template.

11. A polymerase chain reaction method comprising the steps of:

a1) preparing a PCR template according to the method of claim 10;

12. Use of a method for amplifying a nucleic acid according to any one of claims 1 to 9 for performing a polymerase chain reaction.

13. Use of the method for amplifying a nucleic acid according to any one of claims 1 to 9 for detecting a single nucleotide polymorphism.

14. Use of a method for amplifying a nucleic acid according to any one of claims 1 to 9 for detecting a gene mutation.