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PCR (polymerase chain reaction) walking technology and kit used in same

A kit and walking technology, applied in the fields of genetics and molecular biology, can solve the problems of difficulty in ensuring sequence amplification specificity, poor sequence amplification specificity, and difficult control of PCR contamination, etc. The effect of shortening the experiment time

Active Publication Date: 2013-09-04
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as many as 11 semi-random primers are used in the introduced method, and the workload is heavy; reagents are added in the middle of PCR amplification, and the target fragment must be obtained through two rounds of PCR, and there is a low-temperature annealing step in the PCR program, so PCR contamination is difficult Control, sequence amplification specificity is difficult to guarantee
[0014] 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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  • PCR (polymerase chain reaction) walking technology and kit used in same
  • PCR (polymerase chain reaction) walking technology and kit used in same
  • PCR (polymerase chain reaction) walking technology and kit used in same

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 invention discloses semi-random primers, a kit and a method for carrying out PCR (polymerase chain reaction) walking by utilizing the kit. The semi-random primers and the kit are used for the PCR walking technology. The sequence of the semi-random primers are shown in SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7 in a sequence table. The kit comprises the semi-random primers. The method is similar to PCR of the two common primers, can realize target sequence obtainment by only needing two rounds of PCR at most, and is simple, quick and efficient to operate. The method is beneficial to improving the amplification specificity by improving the annealing temperature in the whole PCR cycle process, has no restriction with respect to experiment materials, and is available to microorganism, animal and plant samples. The method can be conductive to simplifying the operation, shortening the experiment time, improving the experiment efficiency and reducing the experiment cost when used in the experiments, thereby having very extensive application prospects.

Description

technical field [0001] The present invention relates to a PCR walking technique and a kit used therefor, in particular to a PCR walking technique and a kit used for a known DNA fragment flanking an unknown sequence, which can be widely used in genetics and molecular biology field. Background technique [0002] 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. [0003] Gene walking technology mainly has the following seven applications: [0004] (1) Isolate flanking sequences of known genes; [0005] (2) Identify the gene insertion site; [0006] (3) Separating and detecting the target gene according to the known fragments of the gene; [0007] (4) for the construction of artificial chromosome fragments to overlap; [0008] (5) constructing contig fragments in map-based cloning; [0009] (6) Transformation of STS or SCAR markers in marke...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 张红发任婧顾瑾麟
Owner BRIGHT DAIRY & FOOD CO LTD