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Multipled PCR detection for deletion type alpha globin gene

A technology of α-globin and deletion type, which is applied in the field of multiple PCR detection, can solve the problems of low efficiency, long PCR running time, large fragments, etc., and achieve good repeatability and stability

Inactive Publication Date: 2005-06-15
深圳益生堂生物企业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Shaji RV et al. Simultaneous detection of -α using multiplex PCR technology 3.7 , -α 4.2 and-- SEA Three kinds of deletions, but the amplified fragments are too large, and the PCR run time is long and the efficiency is not high (Shaji RV, 2000)

Method used

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  • Multipled PCR detection for deletion type alpha globin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The process of detecting deletion α-thalassemia includes the following steps in sequence:

[0032] (1) PCR amplification

[0033] In a sterilized 0.2 or 0.5ml Eppendoef tube, in a 25μl reaction system, add:

[0034] 7 PCR primers at equal concentrations:

[0035] P1: 5'-CCCTCGCCAAGTCCACCCC-3'

[0036] P2: 5'-CAAAGCACTCTAGGGTCCAGC-3'

[0037] P3: 5'-TTTACCCATGTGGTGCCTCC-3'

[0038] P4: 5'-CCGTTGGATCTTCTCATTTCCCC-3'

[0039] P5: 5'-AGCGATCTGGGCTCTGTGTTC-3'

[0040] P6: 5'-CCCACGTTGTGTTCATGGCTG-3'

[0041] P7: 5'-GACCAGGAAGGGCCGGTGC-3

[0042] 7 μl of the mixed stock solution.

[0043] 6 μl of mixed stock solution of four deoxynucleotides dATP, dUTP, dCTP and dGTP at equal concentrations.

[0044] The reaction buffer is 10μl, and its components are 20mmol / L Tris-HCl pH8.9, 50mmol / L KCl, 1.5mmol / L MgCl 2 .

[0045] 30 μl of paraffin.

[0046]Add 1 μl of human genomic DNA, 1 μl of enzyme for PCR reaction, perform PCR cycle after brief centrifugation. The PCR cyc...

Embodiment 2

[0049] The detection process of performing deletion α-thalassemia screening on multiple DNA samples at the same time includes the following steps in sequence:

[0050] (1) PCR amplification

[0051] In a sterilized 0.2 or 0.5ml Eppendoef tube, in a 25μl reaction system, add: equal concentrations of 7 PCR primers:

[0052] P1: 5'-CCCTCGCCAAGTCCACCCC-3'

[0053] P2: 5'-CAAAGCACTCTAGGGTCCAGC-3'

[0054] P3: 5'-TTTACCCATGTGGTGCCTCC-3'

[0055] P4: 5'-CCGTTGGATCTTCTCATTTCCCC-3'

[0056] P5: 5'-AGCGATCTGGGCTCTGTGTTC-3'

[0057] P6: 5'-CCCACGTTGTGTTCATGGCTG-3'

[0058] P7: 5'-GACCAGGAAGGGCCGGTGC-3

[0059] 7 μl of the mixed stock solution.

[0060] 6 μl of mixed stock solution of four deoxynucleotides dATP, dUTP, dCTP and dGTP at equal concentrations.

[0061] The reaction buffer is 10μl, and its components are 20mmol / L Tris-HCl pH8.9, 50mmol / L KCl, 1.5mmol / L MgCl 2 .

[0062] 30 μl of paraffin.

[0063] Add 1 μl of genomic DNA mixture of multiple people (2-6 people) (the ...

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Abstract

A multiple PCR detection method for the depletion-type human alpha-globin gene features that 4 pairs of 7 primers P1, P2, P3, p4, P5, P6 and P7 are used to form quadriplex PCR reaction. The design scheme of said primers and the explanation of result are also disclosed. It can be used for screening and diagnosing three types of alpha-Mediterranean anemia.

Description

technical field [0001] The invention relates to a multiple PCR detection method for medical detection of human α-globin gene. Background technique [0002] The α-globin gene is located in the α-globin gene cluster at the end of the short arm of chromosome 16. The gene cluster includes two repeated α genes (α2 and α1), an embryonic α gene (ξ2), three pseudogenes (ψξ1, ψα2, ψα1) and a gene of unknown function (θ1). It is: 5'-ξ2-ψξ1-ψα2-ψα1-α2-α1-θ1-3', the full length is 50kb (Buckle VJ, 1988; Lauer J, 1980; Michelson AM, 1983; Hatton CS, 1990). Among them, the ξ gene encodes the ξ globin chain, which is expressed in the early fetus. From late fetal period to after birth, ξ is gradually replaced by α2, α1. [0003] The α2 and α1 genes are contained in two highly homologous repeat units consisting of three homologous segments (X, Y, and Z boxes) separated by three non-homologous regions (I, II, and III). Separation (Fig. 2A) (Higgs DR, 1989). During meiosis, the mismatch a...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12Q1/68
Inventor 李泽松耿永尧张文
Owner 深圳益生堂生物企业有限公司
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