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Method for Elution and Sorting of Nucleic Acid Hybrid Molecules

A technology of nucleic acid hybridization and hybridization molecules, applied in the field of sorting microsatellite DNA fragments

Inactive Publication Date: 2011-12-14
XIAMEN UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these cannot be accomplished by traditional elution methods alone

Method used

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Experimental program
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Effect test

Embodiment Construction

[0050] Taking the sorting of TG type microsatellite DNA fragments as an example, the implementation steps of the elution sorting method for nucleic acid hybrid molecules are:

[0051] 1) hybridization. The genomic DNA was digested with the restriction endonuclease Sau3AI, and the corresponding AFLP linker was connected to form an AFLP fragment. In a 50 μL system, 300 ng of AFLP fragments were hybridized with 15 pmol of 5'-end biotin-labeled DNA probes. The probe sequence is: 5'-ATACACAC ACACACACAC ACACACACAC ACACACACACACACACACAC-3' to capture the AFLP fragment containing the TG repeat sequence. The hybridization was carried out in a thermal cycler, and the program was: keep at 95°C for 10 minutes, and lower the temperature by 0.3°C every minute until 37°C. Hybridization products were purified using a DNA recovery kit to remove excess probes.

[0052] 2) Capture. 100 μL of the system contains the hybridization product of step 1), 6×SSC, 0.1% SDS, and 0.01 mg of streptavidin...

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Abstract

The invention relates to an elution sorting method for nucleic acid hybrid molecules, relating to a sorting method for nucleic acid fragments, in particular to a sorting method mainly used for separating microsatellite DNA fragments with different repetition times. Provided is an elution and sorting method for nucleic acid hybrid molecules that can improve the specificity of target nucleic acid fragments, estimate the melting point of hybrid molecules, sort nucleic acid fragments with different matching degrees, and take into account yield and specificity. The nucleic acid capture probe containing the characteristic sequence and the nucleic acid fragment to be sorted are hybridized, fixed on a solid phase substrate, and rinsed with an eluent to obtain a product. By gradually increasing the elution strength of the eluent, more and more specific products are obtained. Analyze the specificity of products under different elution intensities, so as to collect products with different specificities separately, and realize the sorting of similar nucleic acid fragments.

Description

technical field [0001] The present invention relates to a sorting method of nucleic acid fragments, in particular to a sorting method mainly used for separating microsatellite DNA fragments with different repetition times. Background technique [0002] In molecular biology research, it is often necessary to use nucleic acid probes containing specific sequences to hybridize with nucleic acid fragments to be sorted to capture nucleic acid fragments containing their complementary sequences, and to elute under certain conditions to obtain qualified the destination fragment. For the nucleic acid fragments captured by the probes on the solid-phase matrix, in general, pre-eluting with an eluent with low denaturing ability (low temperature, high ionic strength) is used to remove the fragments that cannot be hybridized by the probe but are adsorbed on the solid phase. The nucleic acid fragments on the substrate are then eluted to recover the nucleic acid fragments hybridized on the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C07H21/04
Inventor 曾华嵩丁少雄王颖苏永全
Owner XIAMEN UNIV