Method for determining 5' end flank unknown sequence by using hairpin structure mediation

A technology of hairpin structure and unknown sequence, applied in the field of bioengineering, can solve the problems of inability to determine the sequence of upstream and downstream regulatory elements, unstable mRNA, unsatisfactory effect, etc., and achieve the effect of saving labor, low cost, and less operation restrictions

Inactive Publication Date: 2011-08-03
JIANGNAN UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The RACE method can only obtain the transcribable sequence part of the target gene, but cannot determine the sequence of its upstream and downstream regulatory elements
In addition, the steps of this method are cumbersome, the mRNA is extremely unstable, and reverse transcription is particularly susceptible to low abundance and high-order structures, so the effect is not very ideal
However, the method of using isotope or biotin-labeled probes to hybridize genome or DNA library is time-consuming and labor-intensive.

Method used

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  • Method for determining 5' end flank unknown sequence by using hairpin structure mediation
  • Method for determining 5' end flank unknown sequence by using hairpin structure mediation
  • Method for determining 5' end flank unknown sequence by using hairpin structure mediation

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Effect test

Embodiment 1

[0029] Determination of the Aspergillus usamii E001 xyn10A promoter sequence.

[0030] (1) Preparation of DNA digestion products: PstI was selected for single digestion of Aspergillus usami genomic DNA. The following enzyme digestion system was constructed: 10×H Buffer 2 μl, PstI 2 μl, genomic DNA 10 μl, sterile water 6 μl; the above system was reacted in a 37°C water bath for 4 hours. The purified product (20 μl) was named xyn10AP.

[0031] (2) Preparation of the hairpin structure: HSO (100 μmol / L) 2 μl, sterile water 8 μl, mix well; denature at 94°C for 3 minutes, slowly cool down (1°C / s) to 25°C and then anneal at constant temperature for 30 minutes.

[0032] (3) Ligation of xyn10AP and hairpin structure: 1ul of 10×T4 DNA Ligase Buffer, 3 μl of HSO, 5 μl of xyn10AP, 1 μl of T4 DNA Ligase; overnight at 16°C.

[0033] (4) The first round of PCR amplification of xyn10A promoter: 10×PCR Buffer 2.5 μl, dNTP 1.5 μl, HSO linker 2 μl, HSO-F 0.5 μl, Xyn10A-R1 0.5 μl, sterile water...

Embodiment 2

[0037]Determination of the Aspergillus usamii E001 xyn11A promoter sequence.

[0038] (1) Preparation of DNA digested products: MspI was used to digest the genomic DNA of Aspergillus usami. The following enzyme digestion system was constructed: 2 μl of 10×T Buffer, 2 μl of MspI, 2 μl of 0.1% BSA, 10 μl of genomic DNA, and 4 μl of sterile water; the system was reacted in a water bath at 37°C for 4 hours. The purified product (20 μl) was named xyn11AP.

[0039] (2) Preparation of hairpin joints: HSO (100 μmol / L) 2 μl, sterile water 8 μl, mix well; denature at 94°C for 3 minutes, slowly cool down (1°C / s) to 25°C and anneal at constant temperature for 30 minutes.

[0040] (3) Ligation of xyn11AP and hairpin structure: 1 μl of 10×T4 DNA Ligase Buffer, 3 μl of HSO, 5 μl of xyn11AP, 1 μl of T4 DNA Ligase; overnight at 16°C.

[0041] (4) The first round of PCR amplification of xyn11A promoter: 10×PCR Buffer 2.5 μl, dNTP 1.5 μl, HSO linker 2 μl, HSO-F 0.5 μl, Xyn11A-R1 0.5 μl, steril...

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Abstract

The invention relates to techniques of restriction enzyme cutting, hairpin structure connection, nest type PCR (Polymerase Chain Reaction) amplification and the like, in particular to a method for determining 5' end flank unknown DNA (Deoxyribonucleic Acid) sequence based on a known DNA sequence. The method comprises the following steps of: synthesizing an oligonucleotide sequence easily forming a hairpin structure; selecting a single-restriction enzyme cutting gene group DNA and then connecting the purified single-restriction enzyme cutting gene group DNA with the hairpin structure; selecting a section of specificity sequence on the hairpin structure as the 5' end primer and two complementary sequences on the known DNA sequence close to an unknown sequence end as the 3' end primer by using primer designing software; carrying out nest type PCR amplification by using the hairpin structure connection as a template and adopting the designed primers to acquire the 5' end flank unknown sequence of the known DNA sequence. The method has the characteristics of simple and convenient operation, wide application range, less operation restriction, strong primer specificity, simple and direct result verification, no restriction on the length of the unknown sequence, low cost and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to technologies such as restriction endonuclease (Restriction Endonuclease) digestion, hairpin structure (Hairpin Structure) connection and nested PCR (Nest Polymerase Chain Reaction) amplification, based on known DNA (Deoxyribonucleic Acid) sequence determination of its 5' flanking unknown DNA sequence method. Background technique [0002] With the development of genetic engineering technology and the Internet, more and more gene sequences have been published and can be used by the majority of scientific researchers, but a considerable part of the reported genes have only obtained partial DNA sequences. When we do not know the complete DNA sequence of a gene, we cannot conduct in-depth studies on the structure and function of the gene. In molecular biology and microbial breeding research, it is often necessary to clone flanking regions of known DNA sequences, such as isolatin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邬敏辰汪俊卿张慧敏唐存多高金湖李剑芳吴静
Owner JIANGNAN UNIV
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