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Method for constructing carrier containing klenow-fragment reverse complementary sequences

A reverse complementary, large fragment technology, applied in the field of molecular biology, can solve the problems of time-consuming and laborious, no positive clone growth, low success rate, etc., and achieve the effect of strong operability, great application value and short time.

Pending Publication Date: 2020-06-30
深圳市泽龙生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is time-consuming and laborious, and the success rate is extremely low, and there are often cases where no positive clones grow after inserting the second fragment
On the other hand, the method of homologous recombination is used, because the presence of reverse homologous sequences affects the process of homologous recombination

Method used

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  • Method for constructing carrier containing klenow-fragment reverse complementary sequences
  • Method for constructing carrier containing klenow-fragment reverse complementary sequences
  • Method for constructing carrier containing klenow-fragment reverse complementary sequences

Examples

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Embodiment 1

[0038] This embodiment provides a method for constructing a vector containing a large fragment reverse complementary sequence, comprising the following steps:

[0039] 1. Segmentation containing large fragments of reverse complementary sequences: as attached Figures 1A-1B As shown, the fragment (target fragment 1: SEQ ID NO.1) that needs to be constructed in this embodiment contains a 516bp reverse complementary sequence ( Figure 1A The two underlined parts in the middle, the part in bold font in the middle is a non-complementary sequence, and the length of each underlined part is 516bp). First we split the sequence into two fragments (F1-1 (SEQ ID NO.2) and F1-2 (SEQ ID NO.3) without reverse complementary sequences, Figure 1B The sequence between the underlined part is the target sequence). These two fragments were synthesized separately in a gene synthesis company in Suzhou, and BsaI restriction sites were added to both ends of the fragments (attached Figures 1A-1B , ...

Embodiment 2

[0057] This embodiment provides a method comprising a large fragment reverse complementary sequence vector, comprising the following steps:

[0058] 1. Segmentation containing large fragments of reverse complementary sequences: as attached Figure 4A and 4B As shown, the fragment to be constructed (target fragment 2: SEQ ID NO.5) contains a 609bp reverse complementary sequence. First we split the sequence into two fragments without reverse complement (F2-1: SEQ ID NO.6 and F2-2: SEQ ID NO.7, see Figure 4B , wherein F2-1 includes a structure without a complementary sequence, and the F2-2 sequence has a corresponding complementary sequence in F2-1). These two fragments were synthesized separately in a gene synthesis company in Suzhou, and BmsBI restriction sites were added to both ends of the fragments ( Figure 4B ) contains the target fragment 2 of the reverse complementary sequence of the large fragment. Divide the target sequence into two segments, and add BmsBI restric...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a method for constructing a carrier containing klenow-fragment reverse complementary sequences. The method comprisesthe steps of diving an objective sequence containing the klenow-fragment reverse complementary sequences into two fragments (a fragment 1 and a fragment 2) without reverse complementary sequences; performing gene synthesis on the two fragments, and adding TypeIIS restriction enzyme cutting sites to two ends of each fragment; and enabling complementary sticky terminals to be produced among the fragment 1, the fragment 2 and an objective carrier after the enzyme cutting, and performing connection and transformation so as to obtain positive clone containing the klenow-fragment reverse complementary sequences. The method can effectively solve the difficult problem of constructing the carrier containing the klenow-fragment reverse complementary sequences, and has important significance.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing a vector containing a large fragment reverse complementary sequence. Background technique [0002] Common molecular biology experiments often involve the construction of DNA plasmid vectors. In the process of constructing plasmid vectors, target DNA fragments often contain reverse complementary sequences of large fragments (such as constructing long double-stranded RNA precursor vectors) (>100bp), The reverse complementary sequence can form a hairpin structure, which increases the difficulty of vector construction. At present, gene synthesis is one of the important ways to construct DNA vectors. If the synthesized DNA contains large fragments of reverse complementary sequences, it is difficult and unfeasible to perform gene synthesis. To construct this type of vector, a common enzyme digestion / ligation method is usually used, firstly put a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66
CPCC12N15/66
Inventor 刘云杨玲玲
Owner 深圳市泽龙生物技术有限公司