Hairpin structure-mediated method for determining unknown sequence of 3'-end flanking region

A hairpin structure and unknown sequence technology, applied in the field of bioengineering, can solve the problems of uncertain gene upstream and downstream regulatory element sequences, low reverse transcription abundance, high-level structure, mRNA instability, etc., to save manpower, less operation restrictions, low cost effect

Inactive Publication Date: 2011-08-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 upstream and downstream regulatory element sequences of the gene
In addition, the steps of this method are cumbersome, the mRNA is extremely unstable, and reverse transcription is also easily affected by low abundance and high-level structure, so the effect is not very ideal
However, the method of using isotope or biotin-labeled probes to hybridize and screen genomic libraries is time-consuming and labor-intensive.

Method used

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  • Hairpin structure-mediated method for determining unknown sequence of 3'-end flanking region
  • Hairpin structure-mediated method for determining unknown sequence of 3'-end flanking region
  • Hairpin structure-mediated method for determining unknown sequence of 3'-end flanking region

Examples

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Effect test

Embodiment 1

[0030] Sequence determination of the 3' regulatory region of Aspergillus usamii E001 xyn10A.

[0031] (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 xyn10AT.

[0032] (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.

[0033] (3) Ligation of xyn10AT and hairpin structure: 1 μl of 10×T4 DNA Ligase Buffer, 3 μl of HSO, 5 μl of xyn10AT, 1 μl of T4 DNA Ligase; overnight at 16°C.

[0034] (4) The first round of PCR for the regulatory region of xyn10A 3′ end: 10×PCR Buffer 2.5 μl, dNTP 1.5 μl, HSO linker 2 μl, Xyn10A-F10.5 μl, HS...

Embodiment 2

[0038] Sequence determination of the 3' regulatory region of Aspergillus usamii E001 xyn11A.

[0039] (1) Preparation of DNA digestion products: HindⅢ was used to digest the genomic DNA of Aspergillus usami. The following enzyme digestion system was constructed: 10×M Buffer 2 μl, HindⅢ 2 μl, 0.1% BSA 2 μl, genomic DNA 10 μl, sterile water 4 μl; react the system in a 37°C water bath for 4 hours. The digested and purified product (20 μl) was named xyn11AT.

[0040] (2) Preparation of hairpin joints: 2 μl of HSO (100 μmol / L), 8 μl of sterile water, fully mixed; denatured at 94°C for 3 minutes, slowly cooled (1°C / s) to 25°C and then annealed at constant temperature for 30 minutes.

[0041] (3) Ligation of xyn11AT and hairpin structure: 1 μl of 10×T4 DNA Ligase Buffer, 3 μl of HSO, 5 μl of xyn11AT, 1 μl of T4 DNA Ligase; overnight at 16°C.

[0042] (4) The first round of PCR of xyn11A 3′-end regulatory region: 10×PCR Buffer 2.5μl, dNTP 1.5μl, HSO linker 2μl, Xyn11A-F10.5μl, HSO-R...

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Abstract

The invention discloses a method for determining an unknown sequence of the 3'-end flanking region on the basis of the known DNA sequence, wherein the method relates to the technologies such as restriction enzyme cutting, hairpin structure connection and nested polymerase chain reaction (PCR) amplification. The method comprises the following steps: an oligonucleotide sequence which is easy to form the hairpin structure is synthetized; a single restriction enzyme is selected to perform enzyme cutting to the genomic DNA, the obtained fragments are purified and then connected with the hairpin structure; a primer design software is utilized to select the complementary sequence on the hairpin-structure sequence as 3'-end primer and select two specific sequences on the known DNA sequence near the unknown sequence end as 5'-end primer; and the hairpin-structure connector is used as template, the designed primer is used to perform nested PCR amplification and obtain the unknown sequence in the 3'-end flanking region of the known DNA sequence. The method is characterized by simple operations, wide application range, fewer operational limits, high primer specificity, simple verification, unlimited unknown sequence, low cost and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and is a technique based on known DNA (Deoxyribonucleic Acid) sequence determination of its 3' flanking unknown DNA sequence method. Background technique [0002] With the development of genetic engineering technology and network, more and more gene sequences have been published and can be used by the majority of scientific researchers, but quite a few 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 the process of molecular research, it is often necessary to clone the flanking regions of known sequences, for example, to obtain the regulatory genes of humans, animals and plants according to known genes or molecular markers; to obtain the non-conserved regions of genes in new species through the conserved regions of genes, thereby Obtain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 邬敏辰张慧敏汪俊卿唐存多谭中标李剑芳陈伟
Owner JIANGNAN UNIV
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