Method of introducing a plurality of DNA fragments simultaneously into DNA vector

A fragment and vector technology, applied in the field of genetic engineering, can solve the problems of time-consuming, cumbersome methods, and many limiting factors.

Inactive Publication Date: 2007-08-15
北京华诺奥美基因生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the above-mentioned classical methods are cumbersom

Method used

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  • Method of introducing a plurality of DNA fragments simultaneously into DNA vector

Examples

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Embodiment 1

[0042] Example 1: Cloning the oriT-Am gene cassette and rpsL gene into pBluescript KS(-), while removing the ampicillin resistance gene part of pBluescript KS(-)

[0043] Amplification of the oriT-Am gene cassette with homology arms

[0044] Primer CT1: 5′-

[0045] AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA

[0046] CTGCAGGTCCCCGGGGATCGGTC-3′

[0047] The first 50 bases are part 1801 to 1850 of pBluescript KS(-), and the latter about 23 bases are the 5' end sequence of the amplified oriT-Am gene cassette at 2517-2540 in plasmid pOJ446;

[0048] Primer CT2: 5′-TCAGCCAATCGACTGGCGAGCGGCATCGCA-3′30 bases are 4255-4284 of pOJ446 to amplify the 3′ sequence of the oriT-Am gene cassette; using pOJ446 as a template, a pair of primers CT1 and CT2 were passed through PCR (polymerase chain Reaction) to amplify a 2.0 kb oriT-Am gene cassette with homology arms.

[0049] Amplification of the rpsL gene with homology arms

[0050] Primer CT3:

[0051] 5′-TGCGATGCCGCTCGCCAGTC...

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Abstract

The invention discloses a method to introducing multiple DNA fragment in DNA carrier, which comprises the following steps: augmenting two or more goal DNA fragment with oligonucleotide isogeneic limb through polyose chain-reaction; transforming to permissive state cell with recombinant enzyme vector and armed reforming carrier; getting the reforming carrier with one step through consangnuinity reconstruction of oligonucleotide; making the first fragment 5' end of goal DNA fragment possesses the same sequence with armed replacing position left side of target carrier; setting the last 3' end possesses the same sequence with armed replacing position left side of target carrier; making each fragment 5' end possesses the reversing complementary sequence with the last fragment 3' end from the second fragment. The method of this invention is simple, which can be a general method to reform DNA carrier.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for simultaneously introducing multiple DNA fragments into a DNA carrier. Background technique: [0002] Transforming the existing DNA vectors, such as changing the sequence of the multiple cloning site, introducing one or more foreign DNA fragments, and replacing the original vector part with foreign fragments (such as changing the composition of the resistance gene) is the most important step in molecular biology. One of the basic operations. [0003] The basic steps of the classic DNA vector transformation method are: (1) the original vector and the fragment to be introduced are treated with restriction endonucleases that produce the same sticky ends, or produce complementary sticky ends, or simultaneously produce blunt ends; (2) ) performing agarose gel electrophoresis on two or more target fragments, cutting off the target fragments, and recovering the DNA; (3) c...

Claims

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Application Information

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IPC IPC(8): C12N15/66
Inventor 尚广东谢峰
Owner 北京华诺奥美基因生物科技有限公司
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