Method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I

A technology of topoisomerase and vaccinia virus, applied in the field of connecting DNA fragments, can solve the problems of many steps, cumbersome and time-consuming, etc., and achieve the effects of high connection specificity, wide application range and flexible design

Inactive Publication Date: 2010-03-03
BEIJING TRANSGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method involves many steps and is cumbersome and time-consuming

Method used

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  • Method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I
  • Method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I
  • Method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I

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Embodiment 1

[0029] Two 1.2 kb DNA fragments were ligated using vaccinia virus DNA topoisomerase I.

[0030] 1. Amplification of DNA fragments

[0031] Design primers based on gene sequence:

[0032] Primers 1, 2 and primers 3, 4 each amplify a fragment with a length of 1.2kb, wherein primers 1, 4 only contain sequences complementary to the template, and primers 2, 3 contain adapter DNA sequence, vaccinia The complementary DNA sequence of the viral DNA topoisomerase I recognition site, and the DNA sequence that pairs with the template. The adapter DNA sequences of primers 2 and 3 are reverse complementary.

[0033] The sources, sizes and primers of the two DNA fragments are shown in Table 1.

[0034] The PCR reaction system is 50 μl.

[0035] Lambda DNA template (5ng / μl) 1μl

[0036] 10×TransTaq-T Buffer (with Mg 2+ ) 5μl

[0037] TransTaq-T DNA polymerase (5u / μl) 0.5μl

[0038] 10mM dATP 1μl

[0039] 10mM dGTP 1μl

[0040] 10mM dCTP 1μl

[0041] 10mM dTTP 0.5μl

[0042] 10mM d...

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Abstract

The invention relates to a method for connecting DNA fragments by utilizing vaccinia virus DNA topoisomerase I. The recognition sequence of the vaccinia virus DNA topoisomerase I is introduced into the design of primer; and after being amplified, the DNA fragments are connected by digestion and / or connecting function of the vaccinia virus DNA topoisomerase I. Furthermore, the method is used for constructing TA, UA, blunt end cloning and directional cloning vector, and can be applied to a kit, thus being greatly convenient for the user and providing good technical platform for studies such as gene cloning, expression, protein function and the like.

Description

technical field [0001] The invention relates to a method for connecting DNA fragments by using vaccinia virus DNA topoisomerase I (DNA Topoisomerase I). Background technique [0002] The traditional gene cloning method uses the method of T4DNA ligase (T4DNA Ligase) to connect the target gene to be cloned with the vector, transform the recombinant into the recipient bacteria, and screen and identify the correct clone. However, this method involves many steps and is cumbersome and time-consuming. There is also a method for joining DNA fragments by using the cutting and joining functions of vaccinia virus DNA topoisomerase I. Vaccinia virus DNA topoisomerase I recognizes CCCTT, TCCTT, CCCUU, TCCUU, CCCTU, TCCTU sites. Invitrogen has used the characteristic of vaccinia virus DNA topoisomerase I to recognize the CCCTT site, and after DNA topoisomerase I digestion, different sticky ends have been formed, which have been successfully applied to TA, blunt-end cloning, and directio...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12P19/34
Inventor辛文耿亮张琛李爱玲
OwnerBEIJING TRANSGEN BIOTECH CO LTD