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T-hairpin structure-mediated method for measuring unknown sequence of DNA flank

A DNA sequence, unknown sequence technology, applied in the field of bioengineering, can solve the problems of single connection site, low linker connection efficiency, high price, etc., and achieves the effect of wide application range, labor saving and low cost.

Inactive Publication Date: 2014-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A T carrier-mediated PCR method for determining flanking unknown sequences, the connection is not restricted by restriction endonucleases, and has strong versatility, but the connection efficiency is low with pUCm-T as the linker, and the purchase price of pUCm-T is relatively expensive; A hairpin structure-mediated PCR to determine flanking unknown sequences, and the adapter structure constructed by small molecule nucleotide chains is stable, which greatly improves the specificity and efficiency of PCR amplification, but this method is limited by restriction endonucleases. The connection site is single, which limits its application

Method used

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  • T-hairpin structure-mediated method for measuring unknown sequence of DNA flank
  • T-hairpin structure-mediated method for measuring unknown sequence of DNA flank
  • T-hairpin structure-mediated method for measuring unknown sequence of DNA flank

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Aspergillus usamii (Aspergillus usamii) E001Auseh2 gene 5' flanking unknown DNA sequence determination:

[0030] (1) Construction of HSO-T linker: HSO-T (100 μmol / L) 2 μL, ddH 2 O8 μL, mix well; denature with a PCR gene amplification instrument at 94°C for 3 minutes, slowly cool down (1°C / s) to 25°C, and then anneal at a constant temperature for 30 minutes.

[0031] (2) Preparation of genomic DNA digested products: Xba I was used to digest the genomic DNA of Aspergillus usami. Construct the following digestion system: 10×M Buffer 1 μL, Xba I 0.5 μL, 0.1M BSA 1 μL, genomic DNA 5 μL, ddH 2 O2.5μL; react at 37°C for 4h.

[0032] (3) Enzyme digestion genome modification: 20 μL of enzyme digestion product, 2.5 μL of 10×PCR Buffer, 0.5 μL of dNTP, 0.25 μL of rTaq or Ex Taq, ddH 2O1.75μL; use a PCR gene amplification instrument to react at 72°C for 10min; the modified enzyme-digested product is named Auseh2-DM.

[0033] (4) Connection of Auseh2-DM and HSO-T adapter: 10×T4 ...

Embodiment 2

[0040] Determination of the unknown DNA sequence flanking the 3' end of the Aspergillus usamii E001Auseh2 gene:

[0041] (1) The preparation and connection method of the HSO-T linker and the genomic DNA digestion product are the same as in Example 1.

[0042] (2) Synthesis of upstream primers with known sequences: based on the known Auseh2 gene sequence, two specific downstream primers were synthesized to amplify the unknown sequence flanking the 3′ end of the known DNA sequence, named EH-F1 and EH-F2, Where EH-F2 is more than 30bp away from the 3′ end of the known DNA sequence:

[0043] EH-F1: 5′-TCATGATGCTTGCAAAGCC-3′

[0044] EH-F2: 5′-GGAGAGAAGTTCCTCGAT-3′

[0045] (3) DNA sequence amplification at the 3′ end of Auseh2: 10×PCR Buffer 2.5 μL, dNTP 1.5 μL, THSO-PCR template 2 μL, THSO-F 0.5 μL, EH-F 10.5 μL, ddH 2 O17.75 μL, rTaq enzyme 0.25 μL; 94°C for 5 min, 30 cycles (94°C, 30s; 51°C, 30s; 72°C, 1min20s), 72°C for 10min, the first round of PCR amplification product wa...

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Abstract

The invention relates to a method for amplifying the known sequence of 5' or 3' end flank based on the known DNA sequence by using restriction endonuclease, a T-hairpin structure (HSO-T) and a nest polymerase chain reaction (PCR) amplification technology. The method comprises the following steps: designing an oligonucleotide sequence of the HSO-T; performing enzyme digestion on genome DNA by using single restriction endonuclease and modifying by rTaq or EX Taq; connecting an HSO-T joint with the modified genome; synthesizing a primer THSO-F according to the HSO-T sequence and synthesizing two downstream primers or two upstream primers according to the known DNA sequence; performing nest PCR amplification on the primer THSO-F and the two downstream primers by taking an attachment as a template to obtain 5'-end unknown sequence; and in the same way, performing nest PCR amplification on the primer HSO-F and the two upstream primers to obtain 3'[-end unknown sequence. The technology is simple and convenient to operate, has the advantages of wide application range, low cost, high efficiency, accuracy and the like, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to technologies such as restriction endonuclease (Restriction Endonuclease) digestion, T-Hairpin Structure (T-Hairpin Structure) connection, and nested PCR (Nest Polymerase Chain Reaction) amplification. A method for determining flanking unknown DNA sequences based on known DNA sequences. Background technique [0002] In today's "Gene Wars", the discovery and cloning of new genes has become a research hotspot, and the amplification of unknown flanking sequences plays a pivotal role in molecular biology research. It is the basis of structural genome research and functional genome research. Applied to promoter cloning, obtaining gene arrangement information, determining T-DNA insertion site, chromosome positioning, map-based cloning, gene expression regulation research, artificial chromosome PAC, YAC and BAC fragment overlapping, etc. Currently available methods for determining ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6848
Inventor 邬敏辰汪俊卿胡蝶朱利娟李剑芳
Owner JIANGNAN UNIV
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