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PcMINI vector as well as construction method and application thereof

A construction method and vector technology, applied in the field of molecular biology, can solve the problems affecting RNA splicing efficiency, low efficiency, poor accuracy, etc., and achieve the effects of efficient and rapid vector construction, simple operation and wide application range.

Pending Publication Date: 2021-12-03
武汉翼康基因科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current studies have shown that a large number of human genetic diseases are directly related to abnormal splicing of mRNA: mutations in exon-intron junction sequences can lead to adjacent intron retention or exon deletion; intragene mutations produce new junction sequences Can lead to abnormal RNA splicing; mutations in the sequence of splicing regulatory elements can affect the efficiency of RNA splicing, thereby affecting the level of gene expression
Therefore, how to quickly and efficiently verify abnormal splicing of mRNA in vitro has always been a research hotspot and difficulty. The vector systems reported in the prior art for verifying abnormal splicing of mRNA in vitro have low efficiency and poor accuracy. question

Method used

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  • PcMINI vector as well as construction method and application thereof
  • PcMINI vector as well as construction method and application thereof
  • PcMINI vector as well as construction method and application thereof

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Experimental program
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Embodiment 1p

[0040] The construction of embodiment 1pcMINI vector

[0041] 1.1 Construction of recombinant intermediate vector pcDNA3.1-A

[0042] Using human genomic DNA as a template, use primers pcMINI-HindIII-F and pcMINI-KpnI-R to amplify the "exon A+intron A" DNA fragment; the sequences of the primers are as follows:

[0043] pcMINI-HindIII-F: ACTTAAGCTTatgagtgggctttggggtggccggtt (SEQ ID NO: 5)

[0044] pcMINI-KpnI-R: GCTCGGTACCtacccatgagccatgtg (SEQ ID NO: 6)

[0045] After the above PCR fragments were purified and recovered, the amplified fragments were digested with HindIII / KpnI, and the pcDNA3.1+ plasmid was digested with HindIII / KpnI; Transformed into Escherichia coli DH5α competent cells, and the positive clones screened for ampicillin resistance were double verified by colony PCR detection and sequencing detection, and the recombinant intermediate vector pcDNA3.1-A was obtained.

[0046] 1.2 Construction of pcMINI vector

[0047] Using human genomic DNA as a template, use ...

Embodiment 2

[0052] Example 2 Detection of abnormal mRNA splicing caused by c.1550_1551del mutation of human TSC1 gene

[0053] 2.1 Construction of pcMINI-TSC1 vector

[0054] The c.1550_1551del mutation of the TSC1 gene is located on the No. 15 exon of the TSC1 gene, which will contain part of the No. 14 intron (316bp), No. 15 exon (559bp) and part of the No. 15 intron (304bp) gene fragment Linked into the pcMINI vector to construct the expression plasmid, the map of the expression plasmid is as follows figure 2 shown.

[0055] For the TSC1 wild-type expression vector, using human genomic DNA as a template, use primers TSC1-37525-F / TSC1-39957-R and primers TSC1-37892-F / TSC1-39618-R to carry out nested PCR amplification; the primers The sequence is as follows:

[0056] TSC1-37525-F: gtattctgacttgactatatc (SEQ ID NO: 11)

[0057] TSC1-39957-R: ctgtgttgttagcttaacacag (SEQ ID NO: 12)

[0058] TSC1-37892-F: gtaatgtatgtgggattgctatg (SEQ ID NO: 13)

[0059] TSC1-39618-R: tccccaagcacctgtaa...

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Abstract

The invention discloses a pcMINI vector as well as a construction method and application thereof. The pcMINI vector is formed by modifying a vector pcDNA3.1+, the pcMINI vector comprises a CMV enhancer, a PCMV promoter, a multiple cloning site (MCS), an exon-intron gene fragment and a BGH_PA_terminator terminator, and the multiple cloning site (MCS) is used for being inserted into a gene fragment with mutation at an exon-intron combination position. According to a pcMINI vector system, detection on different gene mutations can be achieved only by replacing inserted DNA fragments, the application range is wide, and the operation is easy and convenient; according to the constructed pcMINI vector, dNA fragments needing to be inserted are smaller, and vector construction is more efficient and faster; and meanwhile, an intron element of the pcMINI vector comprises an efficient splicing acceptor and a splicing donor site, so that effective RNA splicing of an inserted DNA fragment can be ensured. The invention provides a complete and feasible prokaryotic mRNA abnormal splicing verification method based on the pcMINI vector.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a pcMINI vector and its construction method and application. Background technique [0002] In eukaryotes, both proteins and RNA molecules other than mRNA (such as microRNA, cirRNA, lncRNA, etc.) have corresponding coding genes, which are usually separated by non-coding spacers (introns) into Several exons, the above-mentioned coding genes are also called break genes. During the transcription process, the broken gene is first transcribed into a precursor messenger RNA (pre-mRNA) containing exons and introns, and then the precursor messenger RNA (pre-mRNA) is then spliced ​​by RNA to splic the introns. Exon RNA parts are cut and connected in an orderly manner, and finally mature mRNA is produced. RNA splicing is an important biological mechanism of gene expression in eukaryotic cells. Correct RNA splicing depends on the correct combination of spliceosome and...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N15/65C12N15/85C12Q1/6858
CPCC12N15/70C12N15/66C12N15/65C12N15/85C12Q1/6858C12Q2531/113C12Q2535/101C12Q2565/125
Inventor 杨国华胡俊陈志毅李婷张德庆袁天立林宝仪
Owner 武汉翼康基因科技有限公司
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