阅读说明：本技术 基于pcr技术克隆已知区域旁邻的未知dna序列的引物及方法 (Primer and method for cloning unknown DNA sequence adjacent to known region based on PCR technology ) 是由 王高华 李阳 于 2019-09-09 设计创作，主要内容包括：本发明涉及一种基于PCR技术克隆已知区域旁邻的未知DNA序列的引物及方法。引物包括5’端接头区域、中间简并区域和3’端锚定碱基三个组成部分,其中根据物种碱基的偏好性的不同,针对三种碱基群体分布,设计引物的简并区域。引物的设计结合优化后的PCR扩增反应,特异性强,适应性广,能有效地扩增出已知区域旁邻的未知DNA序列。其操作简便,仅需一轮交错热不对称PCR反应,大大节省了商业成本,增加了扩增效率。本发明采用同源重组的形式克隆PCR扩增产物,使非特异性产物在进行同源重组时可以成功的规避掉。同时,在扩增的目标片段为多条或不清晰时,也依然可以进行同源重组反应,获得完整清晰的目标序列。因此,显著减少了实验投入,提高实验效率。(The invention relates to a primer and a method for cloning unknown DNA sequences adjacent to a known region based on a PCR technology. The primer comprises three components of a 5 'end connector region, a middle degenerate region and a 3' end anchoring base, wherein the degenerate region of the primer is designed according to the distribution of three base populations according to the difference of the preference of species bases. The design of the primer is combined with the optimized PCR amplification reaction, the specificity is strong, the adaptability is wide, and the unknown DNA sequences adjacent to the known region can be effectively amplified. The method is simple and convenient to operate, only one round of staggered thermal asymmetric PCR reaction is needed, the commercial cost is greatly saved, and the amplification efficiency is increased. The invention adopts a homologous recombination form to clone a PCR amplification product, so that a non-specific product can be successfully avoided when homologous recombination is carried out. Meanwhile, when the amplified target segment is multiple or unclear, homologous recombination reaction can still be carried out to obtain a complete and clear target sequence. Therefore, the experiment investment is obviously reduced, and the experiment efficiency is improved.)

1. A primer for cloning an unknown DNA sequence adjacent to a known region based on a PCR technology, which is characterized by comprising a degenerate primer and a joint primer; the degenerate primers comprise degenerate primers SEQ ID NO 1-4 aiming at a base sequence with GC content of more than 60 percent, degenerate primers SEQ ID NO 5-7 aiming at a base sequence with GC content of more than 40 percent and less than 60 percent, and degenerate primers SEQ ID NO 8-11 aiming at a base sequence with GC content of less than 40 percent according to different base preferences of species; the sequence of the joint primer is shown as SEQ ID NO 12-14; the formula is shown in the specification, wherein R is A/G, Y is C/T, M is A/C, K is G/T, S is G/C, W is A/T, H is A/T/C, B is G/T/C, V is G/A/C, D is G/A/T, and N is A/T/C/G.

2. a method for cloning unknown DNA sequences adjacent to a known region based on a PCR technology is characterized by comprising the following steps:

(1) Performing a first round of PCR amplification using known genomic DNA as a template and the degenerate primer of claim 1 and the specific primer GSP1 as primers; the degenerate primer is selected from at least one of SEQ ID NO 1-2, or at least one of SEQ ID NO 3-4, or at least one of SEQ ID NO 5, or at least one of SEQ ID NO 6-7, or at least one of SEQ ID NO 8-9, or at least one of SEQ ID NO 10-11;

(2) Performing second round PCR amplification by using the first round PCR amplification product as a template and using an adaptor primer AdP1 and a specific primer GSP2 as primers; the sequence of the joint primer AdP1 is shown as SEQ ID NO. 12;

(3) Performing third round PCR amplification by using the second round PCR amplification product as a template and the adaptor primer AdP2 and the specific primer GSP3 as primers;

(4) And recovering the third round of PCR amplification product, carrying out homologous recombination reaction to clone the target fragment to a vector, and sequencing to obtain complete unknown sequence information.

3. The method of claim 2, wherein the first round of PCR amplification reaction comprises the following steps: pre-denaturation at 90-95 deg.C for 1-5min, and denaturation at 91-97 deg.C for 1-5 min; (1) specific reaction at 90-96 deg.c for 15-60s, T115-60s and 68-75 deg.c for 1-5min for 3-10 cycles; (2) carrying out degenerate primer hybridization reaction at 90-96 ℃ for 15-60s, at 20-30 ℃ for 2-5min, at 0.1-0.5 ℃/s and at 68-75 ℃ for 1-5 min; (3) staggered thermal asymmetric PCR reaction at 90-96 ℃ for 15-60s, T115-60s, 68-75 ℃ for 1-5min, 90-96 ℃ for 15-60s, 40-48 ℃ for 15-60s, 68-75 ℃ for 1-5min, 8-15 cycles; (4) 2-8min at 68-75 ℃; the T1 is the Tm value of GSP 1.

4. The method of claim 2, wherein the second round of PCR amplification reaction comprises the following steps: 1-5min at 91-97 deg.C; 15-40 cycles of 15-60s at 90-96 deg.C, T215-60s, 1-5min at 68-75 deg.C; 2-8min at 68-75 ℃; the T2 is the Tm value of GSP 2.

5. The method of claim 2, wherein the third round of PCR amplification reaction comprises the following steps: 1-5min at 91-97 deg.C; 15-40 cycles of 15-60s at 90-96 deg.C, T315-60s, 1-5min at 68-75 deg.C; 2-8min at 68-75 ℃; the T3 is the Tm value of GSP 3.

6. The method for cloning an unknown DNA sequence next to a known region based on PCR technology as claimed in claim 2, wherein the sequence of the adaptor primer AdP2 is shown in SEQ ID NO. 13.

7. The method of claim 2, wherein the linker primer AdP2 in the third round of PCR amplification is ligated to the Left sequence of the homologous arm of the vector, and the specific primer GSP3 comprises the Right sequence of the homologous arm at the other end of the vector.

8. The method for cloning an unknown DNA sequence next to a known region based on PCR technology as claimed in claim 7, wherein the sequence of the adaptor primer AdP2 is shown in SEQ ID NO. 14.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a primer and a method for cloning unknown DNA sequences adjacent to a known region based on a PCR (polymerase chain reaction) technology.

Background

The chromosome Walking (Genome Walking) technology is a method for gradually detecting an unknown sequence adjacent to a known sequence or a target sequence in a linear relation with the known sequence from the known sequence in a biological Genome or a Genome library. For model biological species (e.g., human, mouse, nematode, rice, Arabidopsis, etc.) whose genome has been sequenced, flanking sequences of known sequences can be easily found from databases. However, the genomic DNA sequence of most organisms in nature is not known so far, and only chromosome walking technology can be used to know the DNA sequence at both sides of a known region. The traditional chromosome walking method comprises the technologies of inverse PCR, joint PCR, plasmid rescue and the like. Because the traditional method has low efficiency, complicated experiment and poor sensitivity and specificity, a plurality of high-efficiency chromosome walking technologies such as TAIL-PCR (staggered thermal asymmetric PCR), HiTAIL-PCR (high-efficiency staggered thermal asymmetric PCR), FPNI-PCR (integrated fusion primer and nested PCR) and the like are researched in recent years. The technology is mainly applied to the walking of sequences flanking known DNA fragments, the identification of T-DNA insertion sites, the filling of gaps by genome sequencing and the like.

The research of plant molecular biology in China tends to the research of the species of the young and the variety research of the species, the detailed DNA sequence of the species is difficult to obtain due to incomplete genome database, limited data volume and diversity among subspecies, so that the PCR chromosome walking technology is the optimal and optimal technology for solving the last unknown DNA sequence. The main problems of the PCR-based chromosome walking technology are how to design specific primers to amplify unknown regions under the condition of unknown sequence information and how to optimize and design a set of PCR product cloning method to realize efficient cloning. Although some highly efficient chromosome walking techniques such as TAIL-PCR have simple test procedures, high automation, long walking distance, and high amplification specificity, random degenerate primers have limited binding sites, the size of specific products is difficult to control, nonspecific bands are still detectable in 2 nd and 3 rd rounds of amplification, and thus, it is difficult to obtain positive results even when different degenerate primers are used.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a primer and a method for cloning unknown DNA sequences adjacent to a known region based on a PCR technology. Therefore, the problem that the complete sequence of the product is difficult to obtain due to non-specific amplification or unclear amplification bands in the chromosome walking process can be solved, the investment in experiments can be obviously reduced, and the experiment efficiency is improved.

In order to realize the purpose, the invention is realized by the following technical scheme:

The first purpose of the invention is to provide a primer for cloning unknown DNA sequences adjacent to a known region based on a PCR technology, which comprises a degenerate primer and a joint primer; the degenerate primers comprise degenerate primers SEQ ID NO 1-4 aiming at a base sequence with GC content of more than 60 percent, degenerate primers SEQ ID NO 5-7 aiming at a base sequence with GC content of more than 40 percent and less than 60 percent, and degenerate primers SEQ ID NO 8-11 aiming at a base sequence with GC content of less than 40 percent according to different base preferences of species; the sequence of the joint primer is shown as SEQ ID NO 12-14; the formula is shown in the specification, wherein R is A/G, Y is C/T, M is A/C, K is G/T, S is G/C, W is A/T, H is A/T/C, B is G/T/C, V is G/A/C, D is G/A/T, and N is A/T/C/G.

The second objective of the invention is to provide a method for cloning unknown DNA sequences adjacent to a known region based on PCR technology, which comprises the following steps:

(1) Performing a first round of PCR amplification using known genomic DNA as a template and the degenerate primer of claim 1 and the specific primer GSP1 as primers; the degenerate primer is selected from at least one of SEQ ID NO 1-2, or at least one of SEQ ID NO 3-4, or at least one of SEQ ID NO 5, or at least one of SEQ ID NO 6-7, or at least one of SEQ ID NO 8-9, or at least one of SEQ ID NO 10-11;

(2) Performing second round PCR amplification by using the first round PCR amplification product as a template and using an adaptor primer AdP1 and a specific primer GSP2 as primers; the sequence of the joint primer AdP1 is shown as SEQ ID NO. 12;

(3) Performing third round PCR amplification by using the second round PCR amplification product as a template and the adaptor primer AdP2 and the specific primer GSP3 as primers;

(4) And recovering the third round of PCR amplification product, carrying out homologous recombination reaction to clone the target fragment to a vector, and sequencing to obtain complete unknown sequence information.

Further, the first round of PCR amplification reaction procedure is: pre-denaturation at 90-95 deg.C for 1-5min, and denaturation at 91-97 deg.C for 1-5 min; (1) specific reaction at 90-96 deg.c for 15-60s, T115-60s and 68-75 deg.c for 1-5min for 3-10 cycles; (2) carrying out degenerate primer hybridization reaction at 90-96 ℃ for 15-60s, at 20-30 ℃ for 2-5min, at 0.1-0.5 ℃/s and at 68-75 ℃ for 1-5 min; (3) staggered thermal asymmetric PCR reaction at 90-96 ℃ for 15-60s, T115-60s, 68-75 ℃ for 1-5min, 90-96 ℃ for 15-60s, 40-48 ℃ for 15-60s, 68-75 ℃ for 1-5min, 8-15 cycles; (4) 2-8min at 68-75 ℃; the T1 is the Tm value of GSP 1.

Further, the second round of PCR amplification reaction amplification procedure is: 1-5min at 91-97 deg.C; 15-40 cycles of 15-60s at 90-96 deg.C, T215-60s, 1-5min at 68-75 deg.C; 2-8min at 68-75 ℃; the T2 is the Tm value of GSP 2.

Further, the third round of PCR amplification reaction amplification procedure is: 1-5min at 91-97 deg.C; 15-40 cycles of 15-60s at 90-96 deg.C, T315-60s, 1-5min at 68-75 deg.C; 2-8min at 68-75 ℃; the T3 is the Tm value of GSP 3.

Further, the sequence of the adaptor primer AdP2 is shown as SEQ ID NO. 13.

Further, the adaptor primer AdP2 in the third round of PCR amplification is connected with the Left sequence of the homologous arm of the vector, and the specific primer GSP3 comprises the Right sequence of the homologous arm at the other end of the vector.

Furthermore, the sequence of the adaptor primer AdP2 is shown in SEQ ID NO. 14.

1. Primer design idea

The primer of the invention consists of three parts: a 5 'end linker region, an intermediate degenerate region, and a 3' end anchoring base. The degenerate primers are designed according to the distribution of three base populations according to the different preference of species bases, and are divided into: (1) a base sequence of high GC content; (2) a neutral base sequence; (3) a base sequence having a low GC content. Two further degenerate regions were designed for the degenerate region of each population primer: (1) primer RDP (random Degenerate primers) with higher degeneracy and strong randomness; (2) primers ADP (arbitrary default primers) with low degeneracy, poor randomness and strong specificity. RDP is a degenerate region composed of H, B, V, D, N degenerate bases, which contain 3-4 bases, so that the degree of degeneracy is high, the possibility of binding to the side of the target gene is increased, the amplification length is increased, and non-specific amplification is increased; ADP is composed of degenerate bases and A, T, G, C fixed bases, so that the degeneracy is reduced, but the specificity is increased and the nonspecific amplification is reduced. 3' end anchoring base adopts a HiTIAL-PCR four-base mode to reduce non-specific amplification.

according to the invention, 11 degenerate primers and three joint primers are designed according to the distribution of three base populations according to the difference of the bias of species bases, wherein the degenerate primers are totally six types: the six primers LRD-L, LRD-N, LRD-H, LAD-L, LAD-N and LAD-H, which can be mixed with each other to increase the degree of degeneracy. Subsequently, nested PCR is used for amplification, so that two nested adaptor primers AdP1 and AdP2 are designed subsequently, and the primers are common PCR amplification primers.

SEQ ID NO:1 LRD-H1：acgatggactccagacggtcbnvnnnggtt

SEQ ID NO:2 LRD-H2：acgatggactccagacggtcbvnvbnnnggtt

SEQ ID NO:3 LAD-H1：acgatggactccagacggtcabgcvtvcgbasnggtt

SEQ ID NO:4 LAD-H2：acgatggactccagacggtcvgcbtvcgbasnggtt

SEQ ID NO:5 LRD-N：acgatggactccagacggtcnnnnnnnnggtca

SEQ ID NO:6 LAD-N1：acgatggactccagacggtcyrnnygmnnkrnggtca

SEQ ID NO:7 LAD-N2：acgatggactccagacggtcrnnygmnnkrnggtca

SEQ ID NO:8 LRD-L1：acgatggactccagacggtchndnnnggaa

SEQ ID NO:9 LRD-L2：acgatggactccagacggtchdndhnnnggaa

SEQ ID NO:10 LAD-L1：acgatggactccagacggtcwgnagwancawaggt

SEQ ID NO:11 LAD-L2：acgatggactccagacggtcdtahcdathgwnggaa

SEQ ID NO:12 AdP1：gagtttaggtccagcgtccgtcgacgatggactccagacggtc

SEQ ID NO:13 AdP2：tcgacgatggactccagacggtc

Wherein r is a/g, y is c/t, m is a/c, k is g/t, s is g/c, w is a/t, h is a/t/c, b is g/t/c, v is g/a/c, d is g/a/t, and n is a/t/c/g.

2. Design idea of PCR amplification

Because the degenerate region of AD is matched and combined with the vicinity of the known fragment, the possibility of combining the degenerate region of AD with the vicinity of the known fragment is reduced due to the small amount of AD primer, and the amplification effect is reduced; and the non-specific amplification is increased rapidly due to the excessive amount of the AD primer, so that false positive bands generated by subsequent amplification are increased, therefore, the AD primer is about 5 times of the gene specific primer 1(GSP1) in the first round of PCR system, and the last two PCR reactions are nested PCR reactions.

In the first round of PCR reaction, the amplification conditions are divided into three parts: (1) the reaction is carried out for 5-10 cycles in the specific amplification stage, and the annealing temperature in the specific amplification stage is higher and is matched with the Tm value of the corresponding GSP1 primer; (2) annealing and hybridizing, reacting for 2-5min at low temperature, and increasing the possibility of pairing the degenerate region and the template DNA; (3) the thermal asymmetric PCR reaction is staggered, 2 rounds of specific high-temperature reaction and 1 round of non-specific low-temperature reaction are set, and the reaction is carried out for 8-15 cycles. The second round of PCR and the third round of PCR are nested PCR reactions, so that the specificity of amplification is increased, the amplification conditions are simple and convenient, and the staggered thermal asymmetric PCR reaction is not performed any more.

3. optimization idea of target sequence cloning mode

The invention adopts a homologous recombination form to clone a PCR amplification product, a AdP2 primer is added with a homologous arm Left of a pUC linearized vector which is AdP2(HR-L), a GSP3 primer is added with a homologous arm Right of the other end of the pUC linearized vector (the pUC sequence of the linearized vector is shown in SEQ ID NO:27) which is GSP3(HR-R), thus the two ends of the third PCR product are provided with the homologous arms of the pUC linearized vector, and in the homologous recombination process, non-specific amplification products AD-AD and GSP-GSP can be successfully avoided because the homologous arms of the two ends only contain Left or Right ends; the subsequent positive clone product is the target sequence. Experiments show that the connection mode of homologous recombination has a great advantage, PCR products can still be recovered when multiple amplified target fragments are or are unclear, homologous recombination reaction can be carried out even if the recovery amount is low, the real target sequence is cloned, and the complete and clear target sequence can be obtained through PCR detection and sequencing.

SEQ ID NO:14AdP2(HR-L)：ccagtgaattcggatccaagctttcgacgatggactccagacggtc

Therefore, compared with the prior art, the invention has the following beneficial technical effects:

(1) The AD primer comprises three components of a 5 'end connector region, a middle degenerate region and a 3' end anchoring base, wherein the degenerate region of the primer is designed according to the distribution of three base groups according to the difference of the preference of species bases. The design of the primer is combined with the optimized PCR amplification reaction of the invention, the specificity is strong, the adaptability is wide, and the unknown DNA sequence adjacent to the known region can be effectively amplified. The method is simple and convenient to operate, only one round of staggered thermal asymmetric PCR reaction is needed in three rounds of PCR amplification, and the whole PCR amplification, product recovery and cloning can be completed within 9-10h, so that the commercial cost is greatly saved, the investment of manpower and material resources is reduced, and the amplification efficiency is increased.

(2) The invention adopts a homologous recombination form to clone a PCR amplification product, so that a non-specific product can be successfully avoided when homologous recombination is carried out, and a subsequent positive clone product is basically a target sequence. Meanwhile, when the amplified target segment is multiple or unclear, homologous recombination reaction can still be carried out to obtain a complete and clear target sequence. Therefore, the experiment investment is obviously reduced, and the experiment efficiency is improved.

Drawings

FIG. 1 is a schematic diagram showing the known sequence of EjCAL and the position of GSP primer in example 1, wherein the underline mark and the square mark are the GSP primer position.

FIG. 2 is a photograph of agarose gel electrophoresis of DNA amplified by the third round of PCR in example 1, wherein M represents 6000bp, 4000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp DNAmarker.

FIG. 3 is a diagram showing the known sequence of the AaMYB6 promoter and the position of the GSP primer in example 2, wherein the position of the square mark is the position of the GSP primer.

FIG. 4 is a photograph of agarose gel electrophoresis of DNA amplified by the third round of PCR in example 2, wherein M represents DNA markers of 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100 bp.

Detailed Description

the following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in the molecular cloning guidelines (third edition, sambrook, inc.), or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available.