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Semi-random primer based on PCR walking technology, and kit thereof

A technology of semi-random primers and kits, applied in the fields of genetics and molecular biology, can solve the problems of poor sequence amplification specificity and cumbersome operation, and achieve the effects of simple operation, improved experimental efficiency, and increased amplification specificity

Active Publication Date: 2013-11-20
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In short, the currently used gene walking methods all have defects such as cumbersome operation and poor specificity of sequence amplification.

Method used

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  • Semi-random primer based on PCR walking technology, and kit thereof
  • Semi-random primer based on PCR walking technology, and kit thereof
  • Semi-random primer based on PCR walking technology, and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 bovine lactoferrin gene PCR walking

[0043]Design upstream primer 1 (P1-1) and upstream primer 2 (P1-2) according to the 5' flanking region of cow bovine lactoferrin gene, its sequence is shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, this implementation The sequence of the semi-random primer AP1 used in the example is shown in SEQ ID NO.3.

[0044] Genomic DNA of commercially available beef was extracted using DNA isolation reagent for meat and meat products (Takara iotechnology (Dalian) Co., Ltd.) kit, and the first round of PCR amplification was performed with P1-1 and AP1.

[0045] The program of the first round of PCR amplification is: ① 92°C, 6 min; ② 94°C, 30s; ③ 50°C, 30s; ④ 72°C, 30s, and steps ② to ④ are 30 cycles.

[0046] The first round of PCR system includes the following final concentrations of each component: 0.2 μmol / L upstream primer 1, 0.2 μmol / L semi-random primer, 0.2 mmol / L dNTP, 1×PCR buffer, 1.5 mM Mg 2+ , 0.02U / μL Taq DNA pol...

Embodiment 2

[0050] Embodiment 2 bovine lactoferrin gene PCR walking sequencing

[0051] Design upstream primer 1 (P1-1) and upstream primer 2 (P1-2) according to the 5' flanking region of cow bovine lactoferrin gene, its sequence is shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, this implementation The sequence of the semi-random primer AP1 used in the example is shown in SEQ ID NO.3.

[0052] Genomic DNA of commercially available beef was extracted using DNA isolation reagent for meat and meat products (Takara iotechnology (Dalian) Co., Ltd.) kit, and the first round of PCR amplification was performed with P1-1 and AP1.

[0053] The program of the first round of PCR amplification is: ① 95°C, 3min; ② 94°C, 30s; ③ 64°C, 30s; ④ 72°C, 180s, and steps ② to ④ are 45 cycles.

[0054] The first round of PCR system includes the following final concentrations of each component: 3 μmol / L upstream primer 1, 3 μmol / L semi-random primer, 0.2 mmol / L dNTP, 1×PCR buffer, 1.5 mM Mg 2+ , 0.02U / μL Taq...

Embodiment 3

[0058] The PCR walking sequence of embodiment 3 bacteria 16s RNA

[0059] According to the 16s RNA sequence of prokaryotic bacteria, the conserved upstream primer P2 was designed, whose sequence is shown in SEQ ID NO.4. The semi-random primer used in this example is AP2, whose sequence is shown in SEQ ID NO.5.

[0060] Using TaKaRa minibest bacterial genomic dna extraction kit ver.2.0 (Takara Biotechnology (Dalian) Co., Ltd.) bacterial genome extraction kit to extract Lactobacillus plantarum ST-Ⅲ (Lactobacillus plantarum ST-Ⅲ) (Bright Dairy Co., Ltd. Provided by the company) strain genomic DNA, do PCR.

[0061] The PCR program is: ① 94°C, 5 min; ② 94°C, 30s; ③ 52°C, 30s; ④ 72°C, 120s, wherein steps ② to ④ are 40 cycles.

[0062] The PCR system includes the following final concentrations of each component: 0.5 μmol / L upstream primer, 0.5 μmol / L semi-random primer, 0.2 mmol / L dNTP, 1×PCR buffer, 1.5 mM Mg 2+ , 0.02U / μL Taq DNA polymerase and 40ng / μL template. The results of e...

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Abstract

The present invention discloses a semi-random primer based on a PCR walking technology, a kit and a method for performing PCR walking by using the kit. The sequence of the semi-random primer is represented by the SEQ ID NO.7 in the sequence list. The kit comprises the semi-random primer. The method is similar to the ordinary two-primer PCR, at most requires two PCR cycles to obtain a target sequence, and has characteristics of simple, rapid and efficient operation. According to the method, an annealing temperature of the whole PCR cycle process is increased, amplification specificity is increased, the used experimental materials are not limited, and the kit can be used by microorganism samples, animal samples and plant samples. The method can be used in experiments so as to simplify operations, shorten an experiment time, improve experiment efficiency, reduce experiment cost, and provide broad application prospects.

Description

[0001] This application is a divisional application of the following applications: filing date, July 19, 2012; application number, 201210251634.5; title of invention, a PCR walking technique and the kit used therein. technical field [0002] The present invention relates to a kind of semi-random primer used in PCR walking technique and kit thereof, in particular to a kind of semi-random primer used in PCR walking technique of known DNA fragment flanking unknown sequence and the kit used thereof, It can be widely used in the fields of genetics and molecular biology. Background technique [0003] So far, the genome sequences of most species are still unknown, and the simple and easy gene walking technology plays an irreplaceable role in the research of modern molecular biology. [0004] Gene walking technology mainly has the following seven applications: [0005] (1) Isolate flanking sequences of known genes; [0006] (2) Identify the gene insertion site; [0007] (3) Separ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张红发任婧
Owner BRIGHT DAIRY & FOOD
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