Method for inserting site in clone provirus

A technology of insertion site and provirus, applied in the field of life science and biology, can solve the problems of cost, difficulty and limited quantity, and achieve high efficiency.

Inactive Publication Date: 2013-12-25
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This not only requires a lot of tedious operations, but also consumes more precious DNA sample resources that are often hard-won, limited in quantity, and precious

Method used

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  • Method for inserting site in clone provirus
  • Method for inserting site in clone provirus
  • Method for inserting site in clone provirus

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Embodiment Construction

[0029] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0030] A method of cloning a proviral insertion site comprising the steps of:

[0031] Step 1) culture cell line, described cell line is BXH-2 mouse leukemia cell line, cultivates in conventional ASM medium and contains 5% CO 2 Incubate at 37°C in a humid environment;

[0032] Step 2) Genomic DNA was extracted, using the Tiangen Genome Extraction Kit to extract genomic DNA from the collected cells according to the operating instructions, and then the DNA dissolved in ultrapure water was stored at -30°C;

[0033] Step 3) carry out the primer extension reaction (APE-PCR) with adenosine as the end, and the primer extension reaction with adenosine as the end comprises the following steps:

[0034] 3.1) Enzyme digestion, use restriction enzyme mixture Hind III, Pvu II and Xho I (Thermo) to digest different genomic DNA, incubate at 37°C ...

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Abstract

The invention discloses a method for inserting a site in the clone provirus. The method includes cultivating a cell line, extracting genome DNA, performing a primer extension reaction with adenosine serving as a tail end, wherein the reaction comprises digestion, extension, purification, connection, first round polymerase chain reaction (PCR), second round PCR and PCR resultant cloning, and performing searching, comparison and analysis in a basic mouse genome database to obtain side wing provirus inset sequences of the genome DNA sequence through a basic local alignment search tool (BLAST). The method provides a tool used for accurately locating insertion sites of a foreign genetic element efficiently, cheaply and rapidly to insert in another genetic material; and even a virus insertion sites can be separated and analyzed from even trace sample DNA. The method can not only be applied in locating the virus insertion sites in a leukemia cell and searching leukemia related genes , but also play an important role in locating insertion sites of other DNA segments.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and specifically relates to a biotin-streptavidin-based purification, using nano-scale streptavidin magnetic beads, and extending the primer by adding terminal adenosine reaction to improve methods for cloning proviral insertion sites. Background technique [0002] The identification and utilization of the human genome and most of the genome sequences of model organisms has greatly promoted the gradual entry of life science and biotechnology into the era of functional genomes. As more and more biomedical organisms and industrially important species are sequenced, understanding the life information carried by these sequences and how they affect the development of cells, the health and behavior of individuals, and even the evolution of organisms, It is an important interesting and long-term work. [0003] The insertional mutagenesis method has been proved to be a fruitful method in m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 尹斌毛政伟徐中娟
Owner SUZHOU UNIV
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