CN112779323A - Dropping-bucket type walking primer and novel PCR method based on dropping-bucket type primer - Google Patents
Dropping-bucket type walking primer and novel PCR method based on dropping-bucket type primer Download PDFInfo
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Abstract
The invention relates to the technical field of molecular PCR walking, in particular to a bucket type walking primer and a new PCR method based on the bucket type primer, wherein the walking primer comprises three bucket primers DBP, the length of the three bucket primers DBP is 25bp, the 5 'end 12bp and the 3' end 3bp of the three are completely the same, the middle 10bp are mismatched pairwise, the bucket primers can only anneal to a bucket complementary site of a previous round of product at low temperature to form a bucket structure, and a new target single chain containing a specific primer complementary site is synthesized by extension; in a subsequent high annealing cycle, the target single strand is converted into a double-stranded molecule, both ends of which are respectively surrounded by the specific primer and the drip chamber primer, so that the double-stranded molecule is exponentially amplified; the non-target single strand cannot be amplified because it cannot be converted into a double strand surrounded by any primer due to the lack of a complete annealing site of any primer. The invention verifies the feasibility of the method by acquiring the unknown flanks of the known DNA of the Lactobacillus brevis CD0817 and the rice genome step by step.
Description
Technical Field
The invention relates to the technical field of molecular PCR walking, in particular to a dropping funnel type walking primer and a novel PCR method based on the dropping funnel type primer.
Background
Genome walking is an important molecular biology technique that is commonly used to obtain the full-length sequence of a gene of interest and clone flanking unknown sequences that are adjacent to known sequences. The genome walking technology is mainly applied to the following aspects: (1) obtaining a regulatory gene for researching the expression regulation of the structural gene; (2) step one, acquiring a non-conservative region of a gene in a new species so as to obtain a complete gene sequence; (3) identifying the insertion site of T-DNA or transposon, and identifying the insertion site of exogenous gene caused by transgenic technology such as gene gun transgenic method; (4) filling gaps in chromosome sequencing work to obtain a complete genome sequence; (5) segment overlapping of artificial chromosomes PAC, YAC and BAC; (6) finding out new functional gene and strain screening. Genome walking has become an important tool for flanking known sequences in the unknown genomic sequences of most organisms in nature.
The genome walking method for separating flanking sequences mainly comprises the following steps: (1) genome walking technology based on genome library screening; (2) PCR-based genome walking techniques. PCR-based methods can be divided into, by principle: PCR methods relying on enzymatic ligation mediation, such as reverse PCR, vector PCR, etc.; random priming PCR, such as thermal asymmetric staggered PCR, semi-random primed PCR. Although the method successfully amplifies the flanking sequences, the method has the defects of complicated operation process, poor specific amplification effect, low connection efficiency and the like. Aiming at the problems, the invention provides a bucket type walking primer and a new PCR method based on the bucket type primer, which can reduce non-specific target products generated in the genome walking process and improve the amplification efficiency.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a dropping funnel type walking primer and a new PCR method based on the dropping funnel type primer, and solves the problems of complicated operation process, poor specific amplification effect, low connection efficiency and the like in the existing genome walking amplification method for separating flanking sequences.
(II) technical scheme
The invention provides a bucket type walking primer and a new PCR method based on the bucket type primer, which can reduce non-specific target products generated in the genome walking process and improve the amplification efficiency by obtaining the unknown flanks of known sequences based on the mismatching of the walking primer.
In order to achieve the above object, the present invention provides a dropper type walking primer, which is characterized in that: the walking primers comprise three drip chamber primers DBP1, DBP2 and DBP3, the length of each primer is 25bp, 12bp at the 5 'end and 3bp at the 3' end of each primer are completely the same, and the middle 10bp are completely different.
Preferably, the two drip chamber primers can anneal with each other only at low temperature, and the annealing temperature between the two drip chamber primers is 35-40 ℃.
Preferably, the novel PCR method comprises three nested PCR amplification procedures, wherein each PCR is amplified by respectively pairing a chamber primer and a specific primer, three chamber primers are sequentially used in the three nested PCR, the first PCR takes a genome as a template, the second and third reactions take the previous PCR product as a template, the three nested PCR procedures are continuously performed, and the three primers in different chamber primer combinations are sequentially matched with SP1, SP2 and SP3 to form three groups of parallel reactions.
Preferably, the specific primers are 2 groups of gene specific primers SP designed according to the DNA sequences of glutamic acid decarboxylase gene of Lactobacillus brevis CD0817 and hygromycin gene in rice genome, each group comprises 3 nested specific primers SP1, SP2 and SP3, the nested specific primers are respectively applied to the 1 st round, the 2 nd round and the 3 rd round, the length of the specific primers is consistent with that of the dropping funnel primers, and the annealing temperature is similar.
Preferably, the sequence of the drip chamber primer is shown as SEQ ID NO 1-SEQ ID NO 3 in the sequence table, the sequence of the specific primer is shown as SEQ ID NO 4-SEQ ID NO 9 in the sequence table, the primer combination of the first round of PCR is SP1-DBP1-DBP2-DBP3, the primer combination of the second round of PCR is SP2-DBP2-DBP3-DBP1, and the primer combination of the second round of PCR is SP3-DBP3-DBP1-DBP 2.
Preferably, the PCR reaction amplification procedure comprises the steps of:
first round PCR: denaturation at 94 ℃ for 3min, and (5) high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2min, and extension at 25 ℃ for 1 very low stringency cycle: denaturation at 94 ℃ for 30s, annealing at 25 ℃ for 30s, and extension at 72 ℃ for 2 min; 25 high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2min, extension at 72 ℃ for 10 min;
second round PCR: denaturation at 94 ℃ for 3min, and (5) high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2min, and 1 low stringency cycle at 40 ℃: denaturation at 94 ℃ for 30s, annealing at 40 ℃ for 30s, and extension at 72 ℃ for 2 min; 25 high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2 min;
third PCR: the amplification conditions were identical to the second round.
Preferably, the first round PCR reaction system comprises: 1 XMg2+plus LA PCR Buffer II, 1.6mM dNTP mix, template DNA, wherein 10-100ng Lactobacillus brevis CD0817 genomic DNA, 100-1000ng rice genomic DNA, 0.2. mu.M drip chamber primer, 0.2. mu.MSP 1 primer, 2.5U TaKaRa LA Taq, sterile distilled water was added to make up the volume to 50. mu.L.
Preferably, the second round PCR reaction system comprises: 1 XMg2+plus LA PCR Buffer II, 1.6mM dNTP mix, 1. mu.L of the first round PCR amplification product, 0.2. mu.M of any one of the different drip chamber primers from the first round, 0.2. mu.M of SP2 primer, 2.5U of TaKaRa LA Taq, sterile distilled water was added to make up the volume to 50. mu.L.
Preferably, the third round of PCR reaction system comprises: 1 XMg2+plus LA PCR Buffer II, 1.6mM dNTP mix, 1. mu.L of the second PCR amplification product, 0.2. mu.M of a different drip chamber primer from the previous two rounds, 0.2. mu.M of SP3 primer, 2.5U of TaKaRa LA Taq, and sterile distilled waterThe volume of the feet was 50. mu.L.
(III) advantageous effects
The invention provides a dropping bucket type walking primer and a new PCR method based on the dropping bucket type primer, which have the following beneficial effects:
(1) according to the bucket type walking primer and the new PCR method based on the bucket type primer, the unknown flanks of the known sequences are obtained based on mismatching of the walking primer, so that non-specific target products generated in the genome walking process can be reduced, and the amplification efficiency is improved.
(2) According to the bucket type walking primer and the new PCR method based on the bucket type primer, target molecules are enriched through 2-3 rounds of nested PCR, and meanwhile, nonspecific products are eliminated.
(3) The dropping bucket type walking primer and the new PCR method based on the dropping bucket type primer use three dropping bucket primers in sequence respectively in three nested PCRs (namely, the dropping bucket primers used by the three PCR are different). Therefore, three different combinations (DBP1-DBP2-DBP3, DBP2-DBP3-DBP1 and DBP3-DBP1-DBP2) can be formed by the three drip chamber primers, so that 3 parallel walking experiments can be carried out, and compared with the traditional primer partial overlap PCR, the method reduces the requirement amount of the walking primers. 3 parallel reactions are carried out simultaneously, so that on one hand, the success probability of the reactions can be increased; on the other hand, the method can amplify fragments with different lengths, and selects the largest fragment for sequencing, namely, the large fragment can be obtained by one step.
Drawings
FIG. 1 is a schematic diagram of the present invention;
FIG. 2 shows the step of flanking sequence of the glutamic acid decarboxylase gene site of Lactobacillus brevis CD0817, wherein (r) represents the different combination sequence of the drip chamber primers in the three-wheel PCR (r) -DBP1-DBP2-DBP 3; ② DBP2-DBP3-DBP 1; ③ -DBP3-DBP1-DBP 2; lane P represents the electrophoresis result of the first round of PCR amplification product; lane S represents the result of electrophoresis of the second round of PCR amplification products; lane T represents the electrophoresis result of the third PCR amplification product; white arrows represent the target product; lane M represents DL 5,000DNA Marker;
FIG. 3 shows the walking result of rice hygromycin gene flanking sequence, wherein (r) shows the different combination sequence of the drip chamber primers in three PCR rounds, (r) -DBP1-DBP2-DBP 3; ② DBP2-DBP3-DBP 1; ③ -DBP3-DBP1-DBP 2; lane P represents the electrophoresis result of the first round of PCR amplification product; lane S represents the result of electrophoresis of the second round of PCR amplification products; lane T represents the electrophoresis result of the third PCR amplification product; white arrows represent the target product; lane M represents DL 5,000DNA Marker.
Detailed Description
In order to further understand the present invention, the following examples are provided to illustrate a bucket type walking primer and a new PCR method based on the bucket type primer, and the scope of the present invention is not limited by the following examples.
Example 1:
lactobacillus brevis PCR walking
Lactobacillus brevis CD0817, which has been deposited at the chinese type culture collection on 7/9/2018, address: eight-way Lojia mountain in Wuchang district, Wuhan, Hubei province, China center for type culture Collection, CCTCC for short, with the preservation number of CCTCC NO: m2018462.
The 5' end of the Lactobacillus brevis CD0817 glutamate decarboxylase gene is provided with gadASP1, gadASP2 and gadASP3, which are respectively combined with three dropping funnel primers to carry out the following PCR reactions:
the first round of PCR reaction system is: 1 × LA PCR Buffer II (Mg)2+plus); dNTP mix, 1.6 mM; genomic DNA, 10-100 ng; any one of the drip chamber primers, 0.2. mu.M; gadASP1 primer, 0.2 μ M; TaKaRa LA Taq, 2.5U; sterile distilled water was added to make up the volume to 50. mu.L. The three dropping funnel primers are respectively reacted in parallel.
The second round of PCR reaction system is: 1 × LA PCR Buffer II (Mg)2+plus); dNTP mix, 1.6 mM; 1 mu L of first round PCR reaction product; any one of the first round of the drip chamber primers, 0.2 μ M; gadASP2 primer, 0.2 μ M; TaKaRa LA Taq, 2.5U; adding sterile distilled water to make up the volume to 50 mu L; the three tubes are performed simultaneously.
The third PCR reaction system is: 1 × LA PCR Buffer II (Mg)2+plus); dNTP mix, 1.6 mM; 1 mu L of second round PCR reaction product; two front wheel dropping funnel primersDifferent drip chamber primers, 0.2. mu.M; gadASP3 primer, 0.2 μ M; TaKaRa LA Taq, 2.5U; sterile distilled water was added to make up the volume to 50. mu.L, and the three tubes were run simultaneously.
The results are shown in FIG. 2. And performing T-clone sequencing on the amplified products of the second round and the third round of PCR reaction, wherein the results show that the obtained DNA molecules are completely overlapped with the ends of gadA. Due to the application of the nested primers, the amplification product of the second round PCR reaction is slightly larger than that of the third round PCR reaction. More than one band of the target DNA is obtained by amplification, which is caused by the random primers of the first round PCR reaction binding to multiple annealing sites on the genomic DNA.
Example 2:
PCR walking of rice
According to the 5' end design of hyg SP1, hyg SP2 and hyg SP3 in the rice genome, the following PCR reactions are carried out:
the first round of PCR reaction system is: 1 × LA PCR Buffer II (Mg)2+plus); dNTP mix, 1.6 mM; rice genome DNA, 100-1000 ng; any one of the drip chamber primers, 0.2. mu.M; hygSP1 primer, 0.2 μ M; TaKaRa LA Taq, 2.5U; sterile distilled water was added to make up the volume to 50. mu.L, and the three tubes were run simultaneously.
The second round of PCR reaction system is: 1 × LA PCR Buffer II (Mg)2+plus); dNTP mix, 1.6 mM; 1 mu L of first round PCR reaction product; any one of the first round of the drip chamber primers, 0.2 μ M; hygSP2 primer, 0.2 μ M; TaKaRa LA Taq, 2.5U; sterile distilled water was added to make up the volume to 50. mu.L, and the three tubes were run simultaneously.
The third PCR reaction system is: 1 × LA PCR Buffer II (Mg)2+plus); dNTP mix, 1.6 mM; 1 mu L of second round PCR reaction product; 0.2 μ M of a chamber primer different from the first two; hygSP3 primer, 0.2 μ M; TaKaRa LA Taq, 2.5U; sterile distilled water was added to make up the volume to 50. mu.L, and the three tubes were run simultaneously.
The results are shown in FIG. 3. T-clone sequencing is carried out on the amplified products of the second round and the third round of PCR reaction, and the result shows that the obtained DNA molecules are completely overlapped with the hyg ends, for example, more than one target DNA band is obtained by amplification, which is probably caused by the combination of random primers of the first round of PCR reaction and a plurality of annealing sites on the genome DNA.
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Claims (9)
1. A dropping bucket type walking primer is characterized in that: the walking primers comprise three drip chamber primers DBP1, DBP2 and DBP3, the length of each primer is 25bp, 12bp at the 5 'end and 3bp at the 3' end of each primer are completely the same, and the middle 10bp are completely different.
2. The dropper type walking primer according to claim 1, wherein: the two drip chamber primers can anneal with each other only at low temperature, and the annealing temperature is 35-40 ℃.
3. A new PCR method based on a dropping-bucket type primer is characterized in that: the novel PCR method comprises three nested PCR reactions, each PCR reaction is driven by one chamber primer and a specific primer corresponding to the PCR reaction, three different combinations are formed by the three chamber primers according to different use sequences, the first PCR reaction takes a genome as a template, the second and third reactions take the former PCR product as a template, the three nested PCR reactions are continuously carried out, and the three primers in the different chamber primer combinations are sequentially and respectively matched with the specific primers SP1, SP2 and SP3 for use, so that three groups of parallel reactions are formed.
4. The novel PCR method of claim 3, wherein: the specific primers are 2 groups of gene specific primers SP designed according to a glutamic acid decarboxylase gene of Lactobacillus brevis CD0817 and a DNA sequence of a hygromycin gene in a rice genome, each group comprises 3 nested specific primers SP1, SP2 and SP3, the nested specific primers are respectively applied to PCR of the 1 st round, the 2 nd round and the 3 rd round, the length of the specific primers is consistent with that of a dropping funnel primer, and the annealing temperature is similar.
5. The novel PCR method of claim 3, wherein: the sequence of the drip chamber primer is shown as SEQ ID NO 1-SEQ ID NO 3 in the sequence table, the sequence of the specific primer is shown as SEQ ID NO 4-SEQ ID NO 9 in the sequence table, the primer combination of the first round of PCR is SP1-DBP1-DBP2-DBP3, the primer combination of the second round of PCR is SP2-DBP2-DBP3-DBP1, and the primer combination of the second round of PCR is SP3-DBP3-DBP1-DBP 2.
6. The PCR reaction of claim 3, wherein: the PCR reaction amplification program comprises the following steps:
first round PCR: denaturation at 94 ℃ for 3min, and (5) high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2min, and extension at 25 ℃ for 1 very low stringency cycle: denaturation at 94 ℃ for 30s, annealing at 25 ℃ for 30s, and extension at 72 ℃ for 2 min; 25 high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2min, extension at 72 ℃ for 10 min;
second round PCR: denaturation at 94 ℃ for 3min, and (5) high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2min, and 1 low stringency cycle at 40 ℃: denaturation at 94 ℃ for 30s, annealing at 40 ℃ for 30s, and extension at 72 ℃ for 2 min; 25 high stringency cycles at 65 ℃: denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 2 min;
third PCR: the amplification conditions were identical to the second round.
7. The PCR amplification program of claim 6, wherein: the first round PCR reaction system comprises: 1 XMg2+plus LA PCR Buffer II, 1.6mM dNTP mix, template DNA, wherein 10-100ng Lactobacillus brevis CD0817 genomic DNA, 100-1000ng rice genomic DNA, 0.2. mu.M drip chamber primer, 0.2. mu.MSP 1 primer, 2.5U TaKaRa LA Taq, sterile distilled water was added to make up the volume to 50. mu.L.
8. The PCR amplification program of claim 6, wherein: the second round PCR reaction system comprises: 1 XMg2+plus LA PCR Buffer II, 1.6mM dNTP mix, 1. mu.L of the first round PCR amplification product, 0.2. mu.M of any one of the different drip chamber primers from the first round, 0.2. mu.M of SP2 primer, 2.5U of TaKaRa LA Taq, sterile distilled water was added to make up the volume to 50. mu.L.
9. The PCR amplification program of claim 6, wherein: the third round of PCR reaction system comprises: 1 XMg2+plus LA PCR Buffer II, 1.6mM dNTP mix, 1. mu.L of the second PCR amplification product, 0.2. mu.M of a different chamber primer from the previous two, 0.2. mu.M of SP3 primer, 2.5U of TaKaRa LA Taq, sterile distilled water to make up the volume to 50. mu.L.
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| CN110540989A (en) * | 2019-09-09 | 2019-12-06 | 湖北伯远合成生物科技有限公司 | Primer and method for cloning unknown DNA sequence adjacent to known region based on PCR technology |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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