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Method for determining bilateral missing sequence of defined DNA sequence utilizing PCR

A DNA sequence and unknown sequence technology, applied in the field of unknown DNA sequences, can solve the problems of inability to obtain regulatory element sequences, consume a lot of manpower and material resources, low abundance and high-level structures, etc., and achieve simple and reliable result analysis, simplify labor and save The effect of time and money

Inactive Publication Date: 2003-05-21
FUDAN UNIV
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AI Technical Summary

Problems solved by technology

Random sequencing (or shotgun method ShotGun Sequencing) is to obtain the required DNA sequence by randomly determining a large number of DNA sequences and then sorting them with the help of computers. material resources
There is another method called RACE (Rapid Amplification of cDNA Ends), which can help to obtain the full-length mRNA sequence of the target gene under the condition that the partial mRNA (messengerRNA) sequence is known, but RACE cannot obtain the upstream and downstream of a gene. Downstream regulatory element sequences, only coding sequences on functional genes can be obtained
The premise of the success of RACE is that the reaction of reverse transcription of mRNA into cDNA (complementary DNA) should be well controlled, but because mRNA is easy to degrade, the reverse transcription reaction is also particularly susceptible to low abundance and high-order structure.
However, the method of using isotope or biotin-labeled probes to hybridize and screen DNA libraries is more time-consuming and laborious, and is the last choice for researchers when they have to.

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  • Method for determining bilateral missing sequence of defined DNA sequence utilizing PCR
  • Method for determining bilateral missing sequence of defined DNA sequence utilizing PCR

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Embodiment 1

[0030]The amplification of Lip I (Alkaline Lipase I, Penicillium arcum PG37 alkaline lipase I) gene promoter sequence is on the basis of known Lip I DNA sequence, adopts the upstream promoter DNA fragment of the amplified Lip I gene of the present invention (The promoter is located upstream of the gene). Firstly, the 3' end primers P1 and P2 were designed and synthesized according to the known sequence, and P2 was close to the promoter end of LipI. TAQ: 5'-CGGCAC-3' and primer LP 5'-GGATCCCTTCACTTCTCAAGTGC-3' at the junction of the 5' end, use the restriction endonuclease Msp1 whose recognition site is CCGG to digest the PG37 genomic DNA; then TAQ and LP in Heating at 50°C for 5 minutes, annealing at room temperature, and ligation with PG37 genomic DNA digestion product at 25°C for 12 hours, LP will be ligated to the cohesive end of the Msp I digestion site at the 5' end of the unknown sequence to be detected. Using the ligation product as a template and LP and P1 as primers,...

Embodiment 2

[0032] Determination of the Complete Sequence of Archaerhodopsin of Halobacteria halobium Species XZ515 Strain, H.sp.xz515 Strain

[0033] Rhodopsin is a seven-transmembrane protein with light-driven proton pump function. In order to study the reasons for the functional differences between it and other proteins with similar functions, it is necessary to obtain the full-length DNA sequence for comparative biological analysis, in order to expect to obtain the proton pump function of Rhodopsin. Rhodopsin was obtained through purification, and protein sequencing was performed on it. However, due to the closed structure of the protein end, primers designed from protein sequencing could not obtain the full sequence of Rhodopsin DNA, and only a part of the DNA sequence in the middle could be obtained. Its two-terminal sequence can be determined by the present invention. For the N-terminal sequence of Rhodopsin, design primers P1 and P2 through the known DNA sequence in the middle: ...

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Abstract

The present invention relates to a method for defining two-side unknow sequence of the know DNA sequence by utilizing PCR technology, and includes the following steps: enzyme cutting restriclion endonuclease site of know sequence; designing proper PCR primer; making PCR amplification; finally making clone and sequence determination of the obtained fragment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for determining unknown DNA sequences on both sides of the DNA or gene sequence from a part of the known DNA (Deoxyribonucleic Acid) sequence by using PCR (Polymerase Chain Reaction) reaction. Background technique [0002] The characteristic of modern biology is that without knowing the complete DNA sequence of a gene, it is impossible to conduct in-depth research on the structure and function of the gene. However, quite a few of the new genes that have been reported so far have only obtained partial DNA sequences. The problem often encountered in research work is to determine the complete DNA sequence of the gene from the known partial DNA sequence, especially the 5' upstream and 3' downstream DNA sequences including various regulatory elements. The current method of determining the unknown sequence at both ends based on the known sequence is limited, which makes t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 黄伟达王奕然钱志康邬敏辰王维荣
Owner FUDAN UNIV
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