CN118048440A - N-based7Genome walking primer and method of HW-PCR - Google Patents

Abstract

The invention discloses a genome walking primer and a genome walking method based on N ₇ HW-PCR, which are characterized in that 7 bases at the 5' end of a random walking primer pWP of primary PCR are degenerated to be N7, and the rest parts are kept unchanged to obtain a primer ds/tWP; ds/tWP is used in the secondary and tertiary PCR, so that the secondary and tertiary walking primers are not completely matched with the primary walking primer, and further, the non-target product obtained by the primary PCR cannot be effectively amplified in the secondary and tertiary PCR and can be eliminated. Compared with the traditional PCR method, the method has the advantages of less primer consumption, simple operation, high efficiency and the like.

Description

Genome walking primer and method based on N 7 HW-PCR

Technical Field

The invention relates to the technical field of PCR walking, in particular to a genome walking primer and a genome walking method based on N ₇ HW-PCR.

Background

Currently, the whole genomic DNA sequence of most organisms in nature is still unknown. To obtain an unknown flanking sequence of a known region, genomic walking techniques are a very efficient method, which has become a common tool in molecular biology research. Among them, PCR-based techniques have become the mainstream genome walking method because of their simplicity and effectiveness. Existing PCR genome walking methods can be divided into two main categories according to their principle: the first is enzyme-ligation mediated PCR, such as inverse PCR, adaptor PCR, etc.; the second category is random priming PCR, such as thermal asymmetric interlaced PCR, wristwatch PCR, forked PCR, etc. The first type of method (i.e., cleavage-ligation mediated PCR) has various drawbacks in practical applications, such as high genome quality requirements, the need for enzymatic digestion and ligation processes, expensive and time-consuming procedures, etc.

The second method (namely random priming PCR) does not need to carry out enzyme digestion and connection on genome, and has the characteristics of simple operation, low price and the like. However, the existing random priming PCR is mainly based on differential amplification to enrich target fragments. Therefore, in the existing random priming PCR method, non-target background is still unavoidable.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a genome walking primer and a genome walking method based on N ₇ HW-PCR, wherein 7 bases at the 5' end of a random walking primer pWP of primary PCR are degenerated to N ₇, and the rest parts are kept unchanged to obtain a primer ds/tWP; ds/tWP is used in the secondary and tertiary PCR, so that the secondary and tertiary walking primers are not completely matched with the primary walking primer, and further, the non-target product generated by the primary PCR cannot be effectively amplified in the secondary and tertiary PCR and is eliminated, thereby improving the concentration of target DNA in the final product.

To achieve the above technical object, in one aspect, the present invention provides a genome walking primer based on N ₇ HW-PCR, wherein the genome walking PCR reaction primer comprises three nest-specific primers pSP, sSP and tSP, and walking primers pWP and ds/tWP; primer pWP is used for primary PCR reaction, and primer ds/tWP is used for secondary and tertiary PCR reaction; degenerate primer ds/tWP the remainder was identical to the corresponding sequence of pWP except that the 7 bases at the 5' end were degenerate to N ₇.

Specifically, the three nest-specific primers pSP, sSP and tSP have a primer length of 20-30nt and a melting temperature of 58-68 ℃.

Further, the primers pWP and degenerate primer ds/tWP, primer length 25nt, melting temperature 58-68 ℃.

In another aspect, the invention also provides a method for performing genome walking based on the N ₇ HW primer, comprising the following steps:

Step 1, first order PCR reaction

Using genome DNA as a template, and carrying out amplification by pairing a primer pSP and pWP;

The primary PCR reaction liquid contains 4-40ng of microorganism genome or 40-400ng of rice genome, 1 XLA PCR Buffer II Mg ²⁺ Plus, 0.2mM of dNTPs, 0.2 mu M of pSP, 0.2 mu M of pWP and 1U of TAKARA LA TAQ hot-start enzyme, and water is used for supplementing to 20 mu L;

step 2, two-stage PCR reaction

Using the primary PCR reaction product as a template, and carrying out amplification by pairing the primer sSP with ds/tWP;

The secondary PCR reaction solution contained 0.4. Mu.L of primary PCR product, 1 XLA PCR Buffer II Mg ²⁺ Plus, dNTPs each 0.2mM, sSP 0.2. Mu.M, ds/tWP 0.2. Mu.M, TAKARA LA TAQ hot-start enzyme 1U, and was supplemented with water to 20. Mu.L;

Step 3, three-stage PCR reaction

Using a secondary PCR reaction product as a template, and performing amplification by pairing a primer tSP and ds/tWP to obtain a target DNA product which is selectively enriched;

The three-stage PCR reaction solution contained 0.4. Mu.L of the secondary PCR product, 1 XLA PCR Buffer II Mg ²⁺ Plus, dNTPs each 0.2mM, tSP 0.2. Mu.M, ds/tWP 0.2. Mu.M, TAKARA LA TAQ hot-start enzyme 1U, and was supplemented with water to 20. Mu.L.

Specifically, the thermal cycle phase of the primary PCR reaction in step 1 is:

stage 1, carrying out 5 high-temperature cycles at 60 ℃;

stage 2, 1 low temperature cycle is carried out at 25 ℃;

Stage 3, 35 high temperature cycles at 60 ℃.

Specifically, the thermal cycling stages of the secondary and tertiary PCR reactions are:

the PCR stage 1 is carried out for 1 cycle at 60 ℃, and then the annealing temperature of each cycle is reduced by 1 ℃ for 10 cycles;

and 2, after the annealing temperature is reduced to 50 ℃, carrying out 10-20 cycles at the temperature of 50 ℃.

Further, in the above 60℃cycle, 50℃cycle and 25℃cycle, the denaturation temperature was 95℃for 30 seconds; the extension temperature was 72℃for 2min.

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

1. The invention obtains a primer ds/tWP by degenerating 7 bases at the 5' end of a primer pWP of random walking of a first round of PCR to N ₇ and keeping the rest unchanged; the ds/tWP is used in the secondary and tertiary PCR, so that the secondary and tertiary walking primers are not completely matched with the primary walking primer, and further, the non-target product obtained by the primary PCR cannot be effectively amplified in the secondary and tertiary PCR and is eliminated, and compared with the traditional PCR, the method is applicable to most genomes, and has the advantages of small primer consumption, simplicity in operation and the like;

2. The invention adopts three walking primer sets, so that three groups of parallel N ₇ HW-PCRs can be simultaneously carried out in a walking experiment, and compared with the traditional PCR, the success rate of walking can be improved;

3. The method has more openness and flexibility, and a user can design more walking primer sleeves according to the self requirements and the primer design principle provided by the invention; only 1-3 sets of parallel N ₇ HW-PCR can be performed in the walking experiment;

4. The walking distance of the method is up to 5.0kb, which is obviously higher than 3.0kb of the conventional walking method, and compared with the conventional method, the walking efficiency of the method is improved by nearly 70%;

5. The ds/tWP primer designed by the invention can improve the specificity and sensitivity of PCR amplification reaction, thereby being capable of detecting the target DNA sequence more accurately.

Drawings

In order to more clearly illustrate the technical solutions of the disclosed embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below. It is to be understood that the following drawings illustrate only certain embodiments of the present disclosure and are therefore not to be considered limiting of its scope, for the person of ordinary skill in the art may admit to other equally relevant drawings without inventive effort.

FIG. 1 is a schematic overall flow diagram of the method of the present invention;

FIG. 2 shows the result of gadR steps of Lactobacillus brevis CD0817 in the examples of the present invention;

FIG. 3 shows the result of gadA steps of Lactobacillus brevis CD0817 in the examples of the present invention;

FIG. 4 shows the hyg walking result of rice in the example of the present invention.

In fig. 1, a thin solid line and a thin broken line represent a known sequence and an unknown sequence, respectively. The arrows and bold lines represent primer and primer complement, respectively. pWP is the primary random walking primer, ds/tWP is the secondary and tertiary degenerate walking primer, pSP is the outermost primer, sSP is the middle primer, and tSP is the innermost primer.

Detailed Description

In order to facilitate the understanding and practice of the present application, a detailed description of the various steps of the method presented herein will follow, with the understanding that these examples are intended to illustrate the application and are not intended to limit the scope of the application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Examples

The invention provides a genome walking primer based on N ₇ HW-PCR, wherein the genome walking PCR reaction primer comprises three nest-specific primers pSP, sSP and tSP, and walking primer pWP and degenerate primer ds/tWP; the primer pWP is used for primary PCR reaction, and the degenerate primer ds/tWP is used for secondary and tertiary PCR reaction; the primer ds/tWP is identical to the corresponding sequence of pWP except that the degeneracy of 7 bases at the 5' end is N ₇.

Specifically, the three nest-specific primers pSP, sSP and tSP have a primer length of 20-30nt and a melting temperature of 58-68 ℃.

Further, the primers pWP and degenerate primer ds/tWP, primer length 25nt, melting temperature 58-68 ℃.

As shown in fig. 1, the present embodiment also provides a method for implementing genome walking based on the N ₇ HW primer, which includes the following steps:

Step 1, first order PCR reaction

Using genome DNA as a template, and carrying out amplification by pairing a primer pSP and pWP;

The primary PCR reaction liquid contains 4-40ng of microorganism genome or 40-400ng of rice genome, 1 XLA PCR Buffer II Mg ²⁺ Plus, 0.2mM of dNTPs, 0.2 mu M of pSP, 0.2 mu M of pWP and 1U of TAKARA LA TAQ hot-start enzyme, and water is used for supplementing to 20 mu L;

step 2, two-stage PCR reaction

Using the primary PCR reaction product as a template, and carrying out amplification by pairing the primer sSP with ds/tWP;

The secondary PCR reaction solution contained 0.4. Mu.L of primary PCR product, 1 XLA PCR Buffer II Mg ²⁺ Plus, dNTPs each 0.2mM, sSP 0.2. Mu.M, ds/tWP 0.2. Mu.M, TAKARA LA TAQ hot-start enzyme 1U, and was supplemented with water to 20. Mu.L;

Step 3, three-stage PCR reaction

Using a secondary PCR reaction product as a template, and performing amplification by pairing a primer tSP and ds/tWP to obtain a target DNA product which is selectively enriched;

The three-stage PCR reaction solution contained 0.4. Mu.L of the secondary PCR product, 1 XLA PCR Buffer II Mg ²⁺ Plus, dNTPs each 0.2mM, tSP 0.2. Mu.M, ds/tWP 0.2. Mu.M, TAKARA LA TAQ hot-start enzyme 1U, and was supplemented with water to 20. Mu.L.

The thermal cycling process for three rounds of PCR reactions in this example is shown in Table 1 below.

TABLE 1 thermal cycling Process for three rounds of PCR reactions

Note that: in the secondary/tertiary PCR, a cycle of 60℃is first performed, then each cycle is reduced by 1℃and after 10 cycles the annealing temperature is reduced to 50 ℃; finally, the remaining cycles were completed at 50 ℃.

Specifically, primary PCR is driven by pWP paired with specific primer pSP; five high temperature (60 ℃) cycles are first performed, at which time only pSP anneals to its complementary site in the known region and extends to the unknown flanking, increasing the copy number of the target single-stranded DNA; in a subsequent low temperature (25 ℃) cycle, pWP is randomly annealed to some positions of the unknown flanking on the just synthesized single strand, and to many sites on the genomic DNA, a series of target and non-target single stranded DNA is newly synthesized; the 5 'end of the newly synthesized target single strand is pWP, and the 3' end of the newly synthesized target single strand is a complementary site of pSP, so that exponential amplification can be performed in the subsequent 60 ℃ cycle; any newly synthesized non-target single strand cannot be amplified in the remaining high temperature cycle due to the lack of perfect binding sites for any primer; however, due to the complexity of the genome and that low temperature 25 ℃ cycle, three types of non-target molecules are still produced in primary PCR: a first class, non-target molecules produced by pSP; a second class, non-target molecules co-produced by pSP and pWP; third, non-target molecules generated by pWP.

The secondary PCR uses the primary PCR product as a template, driven by primer ds/tWP paired with sSP, and the secondary PCR amplification is performed by touchdown PCR. One cycle was performed at 60 c, after which each cycle was reduced by 1c for 10 cycles, and after the annealing temperature was reduced to 50 c, 10-20 cycles were performed at this annealing temperature. Because ds/tWP and pWP have mismatch at the 5' end, the third class of first-stage PCR non-target products (the two ends are pWP) are annealed in the annealing process through the end-mediated molecule, and form a hairpin structure preferentially, so that the third class of first-stage PCR non-target products are prevented from being combined with ds/tWP; the target molecule can be amplified normally through sSP to provide a single chain for ds/tWP annealing; thus the target molecule can be amplified in a near exponential amplification manner; the first class and the second class of first-class PCR non-target products can be easily eliminated through sSP; the landing PCR adopts high Wen Kuozeng at first, so that the amplification stringency is ensured; after the number of target products is increased, the amplification temperature is gradually reduced, and the amplification efficiency is improved.

The third-stage PCR takes a second-stage PCR product as a template and is driven by pairing of a primer ds/tWP and tSP; the working mechanism of the three-stage PCR is consistent with that of the two-stage PCR, and the selective enrichment of target DNA is finally realized.

In order to verify the feasibility and applicability of the method of the invention, the flanking unknown sequences of the gadR, gadA genes in Lactobacillus brevis CD0817 and hyg genes in rice were identified by using the method in this example.

In order to improve the success rate and the walking efficiency of the N ₇ HW-PCR, the invention designs three different walking primer sets (WP 1, WP2 and WP 3) at the same time, wherein each WP primer set consists of pWP and a corresponding ds/tWP primer. A nested set of SPs was designed for each gene (pSP, sSP, tSP). Three WP primer sets were paired with each SP primer set (see Table 2 below) for nested amplification, respectively.

TABLE 2 primers used in the present invention

Note that: a nested set of SP (pSP, sSP, tSP) was designed on the gadR, gadA and hyg genes for primary, secondary and tertiary PCR reactions, respectively. Three WP primer sets were paired with each SP primer set for PCR, i.e., each SP was paired with WP of the same column, respectively, and each ds/tWP was common to both secondary and tertiary PCR.

The amplification results are shown in FIGS. 2-4:

In FIG. 2, tags WP1, WP2 and WP3 represent three parallel N ₇ HW-PCRs performed. Lanes P, S and T represent the primary, secondary and tertiary reactions, respectively, in the N ₇ HW-PCR pool. AS1-AS5 was the gadR secondary N ₇ HW-PCR product. AT1-AT5 is the gadR tertiary N ₇ HW-PCR product. Lane M1 is the TaKaRa DL 5,000DNA marker (5000, 3000, 2000, 1500, 1000, 750, 500, 250 and 100 bp).

In FIG. 3, tags WP1, WP2 and WP3 represent three parallel N ₇ HW-PCRs performed. Lanes P, S and T represent the primary, secondary and tertiary reactions, respectively, in the N ₇ HW-PCR pool. BS1-BS4 was the gadA secondary N ₇ HW-PCR product. BT1-BT4 is gadA tertiary N ₇ HW-PCR product. Lane M1 is the TaKaRa DL 5,000DNA marker (5000, 3000, 2000, 1500, 1000, 750, 500, 250 and 100 bp).

In FIG. 4, tags WP1, WP2 and WP3 represent three parallel N ₇ HW-PCRs performed. Lanes P, S and T represent the primary, secondary and tertiary reactions, respectively, in the N ₇ HW-PCR pool. CS1-CS3 is the hyg secondary N ₇ HW-PCR product. BT1-BT3 is hyg tertiary N ₇ HW-PCR product. Lane M1 is the TaKaRa DL 5,000DNA marker (5000, 3000, 2000, 1500, 1000, 750, 500, 250 and 100 bp).

As can be clearly seen from the figures: clear DNA bands appear in each secondary PCR and tertiary PCR; these bands were recovered by gel cutting and then sequenced; sequencing results show that the target products belong to the same; and the maximum step distance is about 5.0Kb.

In summary, the method of the invention can improve the specificity and sensitivity of PCR amplification reaction by degenerating 7 bases at the 5' end of the primary walking primer pWP to N ₇ and keeping the rest unchanged to obtain the primer ds/tWP, thereby detecting the target DNA sequence more accurately; the ds/tWP is used in the secondary and tertiary PCR, so that the secondary and tertiary walking primers are not completely matched with the primary walking primer, and further, the non-target product obtained by the primary PCR cannot be effectively amplified in the secondary and tertiary PCR and can be eliminated. Compared with the existing method, the method has more universality and universality, can be applied to any genome, can reach 5.0kb of maximum walking distance, is obviously higher than 3.0kb of the conventional walking method, and improves the walking capability by nearly 70%.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims (7)

1. A genome walking primer based on N ₇ HW-PCR, characterized in that the primer of the genome walking PCR reaction comprises three nest specific primers pSP, sSP and tSP, and walking primers pWP and ds/tWP; the primer pWP is used for primary PCR reaction, and the degenerate primer ds/tWP is used for secondary and tertiary PCR reaction; the primer ds/tWP is identical to the corresponding sequence of pWP except that the degeneracy of 7 bases at the 5' end is N ₇.

2. The genome walking primer based on N ₇ HW-PCR according to claim 1, wherein the three nest-specific primers pSP, sSP and tSP have a primer length of 20-30nt and a melting temperature of 58-68 ℃.

3. The genome walking primer based on N ₇ HW-PCR according to claim 1, wherein the primer pWP and degenerate primer ds/tWP have a primer length of 25nt and a melting temperature of 58-68 ℃.

4. A genome walking method using the genome walking primer of any one of claims 1 to 3, comprising the steps of:

Step 1, first order PCR reaction

Using genome DNA as a template, and carrying out amplification by pairing a primer pSP and pWP;

The primary PCR reaction liquid contains 4-40 ng of microorganism genome or 40-400 ng of rice genome, 1 XLA PCR Buffer II Mg ²⁺ Plus, 0.2 mM of dNTPs, 0.2 mu M of pSP, 0.2 mu M of pWP and 1U of TAKARA LA TAQ hot-start enzyme, and water is used for supplementing to 20 mu L;

step 2, two-stage PCR reaction

Using the primary PCR reaction product as a template, and carrying out amplification by pairing the primer sSP with ds/tWP;

The secondary PCR reaction solution contains 0.4 mu L of primary PCR product, 1 XLA PCR Buffer II (Mg ²⁺ Plus), 0.2 mM of dNTPs, 0.2 mu M of sSP, 0.2 mu M of ds/tWP, and TAKARA LA TAQ hot-start enzyme 1U, and the mixture is supplemented to 20 mu L by water;

Step 3, three-stage PCR reaction

Using a secondary PCR reaction product as a template, and performing amplification by pairing a primer tSP and ds/tWP to obtain a target DNA product which is selectively enriched;

The three-stage PCR reaction solution contained 0.4. Mu.L of the secondary PCR product, 1 XLA PCR Buffer II (Mg ²⁺ Plus), dNTPs each 0.2 mM, tSP 0.2. Mu.M, ds/tWP 0.2. Mu.M, TAKARA LA TAQ hot-start enzyme 1U, and was supplemented with water to 20. Mu.L.

5. The genomic walking method of claim 4, wherein the thermal cycling phase of the primary PCR reaction in step 1 is:

Stage 1, 5 cycles at 60 ℃;

stage 2, 1 cycle at 25 ℃;

Stage 3, 35 cycles at 60 ℃.

6. The genomic walking method of claim 4, wherein the thermal cycling process of the secondary and tertiary PCR reactions is:

1 cycle is carried out at the temperature of 60 ℃ in the stage 1, and then the annealing temperature of each cycle is reduced by 1 ℃ for 10 cycles;

and 2, after the annealing temperature is reduced to 50 ℃, carrying out 10-20 cycles at the temperature of 50 ℃.

7. The genomic walking method of claim 5 or 6, wherein the denaturation temperature during the 60 ℃,50 ℃ and 25 ℃ cycles is 95 ℃ for 30 s; the extension temperature was 72℃for 2min.