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Kits and methods for direct nucleic acid amplification from whole blood samples

A whole blood sample and nucleic acid technology, applied in the field of nucleic acid amplification, can solve problems such as cumbersome operation steps

Inactive Publication Date: 2011-12-21
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a reaction kit for nucleic acid amplification using whole blood as a starting template, which solves the problem caused by the need for pretreatment of whole blood in the prior art when whole blood is used as a starting template to directly perform nucleic acid amplification. Problems such as cumbersome operation steps

Method used

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  • Kits and methods for direct nucleic acid amplification from whole blood samples
  • Kits and methods for direct nucleic acid amplification from whole blood samples
  • Kits and methods for direct nucleic acid amplification from whole blood samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～27

[0054] The optimization experiment of embodiment 1~27 PCR amplification reaction system

[0055] This embodiment uses a single factor optimization test to detect the impact of PCR amplification reaction solutions containing different concentrations of tris, magnesium ions, and glycerol on the amplification efficiency; wherein in Examples 1 to 9, the concentrations of Tris bases are respectively controlled at 0, 10, 25, 50, 75, 100, 125, 150 and 200mmol / L, magnesium ion concentration adopts 2.0mmol / L, glycerin concentration is 10%, other conditions are stable; embodiment 10~18 controls magnesium ion concentration respectively At 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0mmol / L; Tris alkali concentration is controlled at 50mmol / L respectively, and glycerol concentration is 10%, and other conditions are stable; Embodiment 19~27 makes glycerol concentration Respectively controlled at 0, 2%, 4%, 6%, 8%, 10%, 12%, 14% and 16%, Tris base concentration was controlled at 50mmol / L, ma...

Embodiment 28

[0065] Example 28 Detection of single nucleotide polymorphisms associated with esophageal cancer

[0066] This example is used to simultaneously determine three single nucleotide polymorphism sites associated with esophageal cancer

[0067] 1. Experimental materials: blood samples treated with different anticoagulants (ethylenediaminetetraacetic acid disodium salt, sodium citrate); genomic DNA, extracted by phenol / chloroform extraction; 9 primer sequences are shown in Table 1, Synthesized by Jinsite Technology (Nanjing) Co., Ltd., PAGE grade; other materials are the same as in Example 1.

[0068] Genomic DNA was extracted using the following methods:

[0069] 1) Sampling: Take 1-2ml of peripheral blood from the patient and place it in sodium citrate (1:9) or EDTA anticoagulant tubes instead of heparin anticoagulant tubes. Aspirate part of the blood in the anticoagulant tube and centrifuge at 8000rpm for 5 minutes to separate plasma and cells. Carefully transfer the plasma i...

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Abstract

The invention discloses a reaction kit for nucleic acid amplification using whole blood as a starting template, which is characterized in that the nucleic acid reaction system of the kit includes several primer pairs for amplifying target sequences of target genes; And the concentration of tris in the system is between 25-100mmol / L, the concentration of magnesium ions is between 1.5-3.0mmol / L, and the concentration of glycerol is between 10%-14%. The kit can efficiently amplify the target nucleic acid from the whole blood sample containing a large amount of substances that inhibit nucleic acid amplification without going through the nucleic acid separation and purification process, and without any pretreatment of the whole blood sample.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and in particular relates to a kit and a method for directly performing nucleic acid amplification from whole blood samples. Background technique [0002] Nucleic acid amplification technology based on polymerase chain reaction (PCR) is a modern molecular biological technology that amplifies a very small amount of nucleic acid samples by millions or even hundreds of millions of times. fields are widely used. Typically, PCR amplification requires genomic DNA as a template, and genomic DNA is extracted from peripheral blood. Currently, methods commonly used to extract DNA from peripheral blood include phenol / chloroform extraction, salting out, potassium iodide, and boiling lysis [Carpi FM, DiPietro F, Vincenzetti S, et al. Human DNA extraction methods: patents and applications. Recent Patents on DNA & Gene Sequences 2011, 5(1): 1-7.] Many commercial DNA extraction kits have bee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 汪维鹏
Owner SUZHOU UNIV
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