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Method and kit for directly amplifying DNA (deoxyribonucleic acid) segment from biological material

A kit and direct technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as difficulty in obtaining and loss, and achieve the effects of convenient operation, safe use, and wide application range

Inactive Publication Date: 2012-01-18
生工生物工程(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For some trace samples, especially some more precious samples, if DNA extraction is performed by conventional methods, a small amount of DNA will be lost during the step-by-step operation, and finally it is difficult to obtain DNA for the next experiment

Method used

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  • Method and kit for directly amplifying DNA (deoxyribonucleic acid) segment from biological material
  • Method and kit for directly amplifying DNA (deoxyribonucleic acid) segment from biological material
  • Method and kit for directly amplifying DNA (deoxyribonucleic acid) segment from biological material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] (1) the solution adopted in embodiment 1

[0084] A. Sample lysate a, the sample lysate formula is

[0085] Lysis solution a-1: 70% PEG200; 20mM KOH, pH 13.5;

[0086] Lysis solution a-2: 60% PEG200; 20mM KOH, pH 13.5;

[0087] Lysis solution a-3: 60% PEG200; 40mM KOH, pH 13.8;

[0088] Lysis solution a-4: 50% PEG200; 20mM KOH, pH 13.5.

[0089] B. Neutralizing solution, the formula is as follows

[0090] 200mM Tris-HCl (pH 6.8-7.5).

[0091] C.2×Taq PCR mixture, the formula is as follows

[0092] 2×PCR Buffer;

[0093] 3mM MgSO 4 ;

[0094] 0.2mM dNTPs;

[0095] 0.1U / μL Taq DNA polymerase;

[0096] 2×Loading Buffer.

[0097] D.2×PCR Buffer, the formula is as follows:

[0098] 40mM Tris-HCl, pH=8.825°C;

[0099] 20mM (NH4) 2 SO 4 ;

[0100] 20mM KCl;

[0101] 0.2%Trtion X-100;

[0102] 0.2mg / mLBSA.

[0103] E.2×Loading Buffer, the formula is as follows:

[0104] 8% Glycerol;

[0105] 0.05% Bromophenol Blue.

[0106] (2) Genomic DNA of tobacco was e...

Embodiment 2

[0119] (1) the solution adopted in embodiment 2

[0120] A. Sample lysate b, the sample lysate formula is

[0121] Lysis solution b-1: 5% chelex-100 (chelex-100 was purchased from sigma company)

[0122] Lysis solution b-2: 10% chelex-100 (chelex-100 was purchased from sigma company)

[0123] B. 2×Taq PCR mixture, the formula is as follows

[0124] 2×PCR Buffer;

[0125] 3mM MgSO 4 ;

[0126] 0.2mM dNTPs;

[0127] 0.1U / μL Taq DNA polymerase;

[0128] 2×Loading Buffer.

[0129] The 2×PCR Buffer and 6×Loading Buffer used therein are exactly the same as those in Example 1.

[0130] (2) Genomic DNA is extracted from a small amount of blood.

[0131] 1. Take 5-10 μL of anticoagulated whole blood and add it to a 1.5ml centrifuge tube, add 500 μl of sterile water, and shake vigorously.

[0132] Centrifuge at 12,000rpm for 2min, discard the supernatant, and collect the precipitate.

[0133] 2. Add 100 μL Lysis Solution 2 to the precipitation, bathe in water at 56°C for 30 m...

Embodiment 3

[0143] (1) the solution adopted in embodiment 3

[0144] A. Sample lysate c, the sample lysate formula is

[0145] Lysis solution c-1: 10% chelex-100, 1% TritonX-100;

[0146] Lysis buffer c-2: 10% chelex-100, 2% TritonX-100.

[0147] B. 2×Taq PCR mixture, the formula is as follows

[0148] 2×PCR Buffer;

[0149] 3mM MgSO 4 ;

[0150] 0.2mM dNTPs;

[0151] 0.1U / μL Taq DNA polymerase;

[0152] 2×Loading Buffer.

[0153] The 2×PCR Buffer and 6×Loading Buffer used therein are exactly the same as those in Example 1.

[0154] (2) Genomic DNA extraction from hair.

[0155] 1. Take 3-4 hairs with hair follicles, take the roots, put them into a 1.5ml centrifuge tube, add 30μl lysate c and 1μL Proteinase K, shake, 56°C water bath for 30min, 100°C water bath for 8min.

[0156] 2. Centrifuge at 12,000 rpm for 3 minutes, and take the supernatant for PCR reaction.

[0157] (3) PCR reaction

[0158] The template obtained above was subjected to PCR amplification. The upstream and...

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Abstract

The invention discloses a method and kit for directly amplifying a DNA (deoxyribonucleic acid) segment from a biological material. The kit disclosed by the invention comprises a lysis solution a, a lysis solution b, a lysis solution c and a neutralization solution, and can also comprise a 2*Taq PCR (polymerase chain reaction) mixed solution, wherein the lysis solution a comprises 50-70v% PEG (polyethylene glycol) 200 and 20-40mM KOH, and the pH value of the lysis solution a is 13.2-13.8; the lysis solution b comprises 5-10wt% chelex-100; the lysis solution c comprises 5-10wt% chelex-100 and 1-2wt% TritonX-100; and the neutralization solution comprises a 190-210 mmol / L Tris-HCl buffer of which the pH value is 6.8-7.5. The kit and method disclosed by the invention are applicable to many samples, such as bacteria, animal tissues, plant tissues, fungi, blood, saliva, hair and the like.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering. Specifically, the present invention relates to a method for amplifying target genomic DNA fragments from trace biological samples. Further, the present invention relates to a kit based on the method, its reagent composition and operation method. Background technique [0002] DNA is the carrier of genetic information and the most important biological information molecule. With the development of molecular biotechnology, genomic DNA extraction technology and PCR amplification technology have become the basis of molecular biology research, and also the most important link in genomic DNA and related research. [0003] DNA exists in the nuclei, mitochondria, and chloroplasts of various organisms, and can also exist in the cytoplasm of some cells in a free state. As an important genetic material basis, DNA has three important characteristics: (1) Consistency of the same indi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 芦丽亚张丽娟李威
Owner 生工生物工程(上海)股份有限公司
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