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Polymerase chain reaction (PCR)-based quick construction method for tandem repetitive sequence and special primer pair and use

A technology of tandem repeat sequences and primer pairs, applied in recombinant DNA technology, DNA/RNA fragments, DNA preparation, etc., can solve the problems of troublesome screening and identification, low recovery efficiency, etc., to achieve fast and convenient identification and screening, and speed up experiments. Speed, effect of simplifying experimental procedures

Inactive Publication Date: 2012-05-23
HUNAN AGRICULTURAL UNIV
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Problems solved by technology

Since the cis-acting element is usually small (less than 100bp), the efficiency of recovery is low, and there will be two situations of forward connection and reverse connection when connecting at the same time, and the probability of more than 3 repeated fragments is very small, only about 2%. Left and right, screening and identification are quite troublesome

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  • Polymerase chain reaction (PCR)-based quick construction method for tandem repetitive sequence and special primer pair and use
  • Polymerase chain reaction (PCR)-based quick construction method for tandem repetitive sequence and special primer pair and use
  • Polymerase chain reaction (PCR)-based quick construction method for tandem repetitive sequence and special primer pair and use

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Embodiment Construction

[0024] The present invention will be further described below.

[0025] The rd29A gene was cloned from drought-treated Arabidopsis by Yamaguchi-Shinozaki et al. It is not only induced by drought, but also induced by high salt and low temperature. The expression of rd29A gene is regulated by DREB transcription factors under drought, high salinity and low temperature stress. A 71 bp DRE cis-acting element (see SEQ ID NO.1 for its sequence) was identified in its promoter sequence, and this cis-acting element contains the core sequence of TACCGACAT.

[0026] 1. Construction of low-copy direct tandem DRE repeats by PCR method:

[0027] Materials: pCAMBIA1301 plasmid containing the 71bp DRE cis-acting element of the rd29A promoter (provided by Professor Chen Shouyi, Institute of Genetics and Development, Chinese Academy of Sciences).

[0028] 1. The design of the primer pair is based on the 71bp DRE cis-acting element sequence of the 29A promoter, and is designed with PrimerPremier...

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Abstract

The invention discloses a PCR-based quick construction method for a tandem repetitive sequence, a special primer pair and use. The method constructs a low-copy tandem repetitive sequence of target cis-acting elements which are in the same direction by performing PCR amplification of the target cis-acting elements by using a primer pair designed according to the target cis-acting elements. In the method, more than 3 tandems in the same direction are quickly constructed by performing only one PCR, the positive cloning rate reaches 18 percent, and 200 tandems in the same direction can be obtained by performing two PCRs and subcloning once. The low-copy trandems are used for the quick and effective construction of repetitive cis elements in yeast crossing, and the obtained high-copy tandems can be used very widely in the production of high-molecular-weight repetitive proteins.

Description

Technical field: [0001] The invention relates to a method for constructing a tandem repeat sequence, in particular to a method for quickly constructing a tandem repeat sequence by PCR and the application of the method in the construction of a promoter core repeat sequence in yeast one-hybrid. Background technique: [0002] The regulation of various transcription factors at the transcription level in eukaryotes is the main way of gene expression regulation. Typical transcription factors have DNA binding regions and transcription activation regions (or repression regions). At specific times, locations or under specific conditions (such as stress conditions), specific transcription factors are specific to the cis-acting elements of the target gene promoter. Sexual binding and interaction can activate or inhibit the expression of target genes. [0003] It is necessary to clarify the mechanisms of various signal transduction and gene expression in cells, as well as a series of s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12N15/113
Inventor 陈己任熊兴耀邓子牛
Owner HUNAN AGRICULTURAL UNIV
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