Enhanced vpr protein and plasma free nucleic acid extraction method

A free nucleic acid, enhanced technology, applied in the field of biomedicine, can solve problems such as nucleic acid degradation, and achieve the effect of reducing nucleic acid degradation, high catalytic activity, and high activity

Active Publication Date: 2020-11-17
BEIJING KEXUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to provide an enhanced VPR protein and a method for extracting plasma free nucleic acid to solve the technical problem in the prior art that proteinase K needs to be heated to cause nucleic acid degradation in the extraction of plasma free nucleic acid

Method used

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  • Enhanced vpr protein and plasma free nucleic acid extraction method
  • Enhanced vpr protein and plasma free nucleic acid extraction method
  • Enhanced vpr protein and plasma free nucleic acid extraction method

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Experimental program
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Embodiment approach

[0062] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecules in the above recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0063] Although the qualifier used in the present invention is "contains" when limiting the above-mentioned DNA molecule, it does not mean that other sequences irrelevant to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or additionally add start codons, stop codons, etc., therefore, if using A closed-ended statement will not truly cover these situations.

[0064] The ...

Embodiment 1

[0070]Prepare buffers for magnetic bead nucleic acid extraction, including lysis buffer, washing buffer and elution buffer. The high-concentration salt composition of the lysis buffer is one or more of the following: 2-3M guanidine hydrochloride, 4.5-6M guanidine isothiocyanate, 1.5-3M sodium iodide, and the like. Other ingredients are: 1%-30% Triton X-100, 10-50mM tris-hydrochloride (Tris-Cl), 1-10mM sodium ethylenediaminetetraacetate (EDTA).

[0071] Component A of the washing buffer solution is: 2-3M guanidine hydrochloride, 1%-30% Triton X-100, 5-50mM tris-hydrochloride (Tris-Cl), 1-10mM Sodium ethylenediaminetetraacetate (EDTA), isopropanol, 70% ethanol.

[0072] Component B of the washing buffer is: 5-50 mM tris-hydrochloride (Tris-Cl), 1-10 mM sodium ethylenediamine tetraacetate (EDTA), and 80% ethanol.

[0073] The composition of the elution buffer is: 8-10 mM Tris hydrochloride (Tris-Cl, pH 8.0).

[0074] The magnetic beads are superparamagnetic nano-magnetic beads...

Embodiment 2

[0088] Comparison of the Activities of Enhanced VPR Protease and Proteinase K at Different Temperatures

[0089] Under 5 temperature conditions (15~55 ℃), measure enhanced VPR protease (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:4 respectively) ID NO:6) and proteinase K (below) Enzyme kinetic parameters for the hydrolysis of the substrate succinyl-AAPF-p-nitroanilide, K cat / K m Values ​​are calculated according to Michaelis-Menten equation and substrate concentration.

[0090] The amino acid sequence of proteinase K is SEQ ID NO: 12:

[0091] AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA

[0092] Enhanced VPR protease (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, SEQ ...

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Abstract

The invention discloses a method for extracting enhanced VPR (viral protein regulatory) protein and plasma free nucleic acid. An amino acid sequence of the enhanced VPR protein is obtained by the mutation of the amino acid sequence shown in SEQ ID NO:1; the mutation at least comprises one of following mutation sites: a 55th site, a 102nd site, a 134th site and a 166th site; the mutation of tyrosine at the 55th site is phenylalanine; the mutation of serine at the 102nd site is threonine; the mutation of serine at the 134th site is isoleucine; the mutation of serine at the 166th site is threonine; or, the amino acid sequence of the enhanced VPR protein has the mutation sites in the amino acid sequence with mutation, and has more than 98% homology with the amino acid sequence with mutation. The enhanced VPR protein has the advantage that the activity is higher at normal temperature, so that the heating is not required in the plasma free nucleic acid extraction process, and the degradationof nucleic acid is reduced.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for extracting enhanced VPR protein and plasma free nucleic acid. Background technique [0002] The extraction of free nucleic acid from plasma is a key experimental step in fetal non-invasive prenatal screening and tumor liquid biopsy, because the total amount of free nucleic acid directly affects the sensitivity and detection limit of non-invasive prenatal screening and tumor liquid biopsy. At present, there are a variety of extraction methods and commercial kits to choose from, which can be roughly divided into two categories: spin column method and magnetic bead method. There are certain differences in the simplicity of experimental operation and the amount of free nucleic acid extracted. The spin column method utilizes a silica-based membrane to bind nucleic acids, centrifuge to remove impurities, and then elute the nucleic acids from the silica-based membrane. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/70C12N1/21C12N15/10
CPCC12N9/50C12N15/101C12N15/1013C12Q2521/537
Inventor 谭泽民白灵方楠刘运超王建伟刘倩唐宇
Owner BEIJING KEXUN BIOTECH CO LTD
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