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Nucleic acid amplification method

A nucleic acid and nucleotide sequence technology, applied in biochemical equipment and methods, DNA preparation, microbial measurement/inspection, etc., can solve the problem of low amplification efficiency, low non-specific products, low amplification efficiency, and increased non-specific products, etc. problem, to avoid the effect of reducing the amplification efficiency

Inactive Publication Date: 2014-03-26
SNOVA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In view of this, the purpose of the present invention is to provide a nicking enzyme-coupled transcription mediator with high amplification efficiency and less non-specific products to address the problems of reduced amplification efficiency and increased non-specific products caused by the addition of RP in the prior art. Guided Nucleic Acid Amplification Method

Method used

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Examples

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Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Respectively use restriction endonuclease and restriction nickase to amplify nucleic acid with different concentrations, compare detection sensitivity and specificity of amplified products

[0088] 1. According to the method of Chinese patent CN02807389.4, use restriction endonucleases to amplify nucleic acids with different concentrations (control group):

[0089] Reaction conditions: A conservative restriction endonuclease XbaI is encoded in the conserved region of S-gen of HBV DNA (according to nt244-285 of the EcoRI site). When this portion of the S-region is capable of becoming single-stranded DNA of negative polarity, oligonucleotides ("restriction primers" (RP)) complementary to the region containing the sequence Forms a double-stranded restriction site. Use the HBV quality control product nucleic acid (including the site to be tested, single-stranded DNA) developed by our company for gradient detection, and use 5 μL of extract for each measurement (a...

Embodiment 2

[0099] Embodiment 2: Amplification of HBV DNA (template is single-stranded DNA)

[0100] A conserved restriction nickase Nt.CviPII is encoded in the conserved region of S-gen of HBV DNA (according to nt257-262 of the EcoRI site, --TGGˇTGGˇA--). When this part of the S-region is capable of becoming single-stranded DNA of negative polarity, a promoter primer is added, in this method a promoter primer is used whose 3' part contains a sequence exactly complementary to the 3' end at the cut site, and The 5' end contains a promoter sequence that can be recognized by RNA polymerase (such as T7). Double-stranded restriction sites are thereby formed for all genomic DNA present. 6 μL of extract was used for each assay. Restriction enzyme digestion was carried out under the following conditions: TMA buffer 50mM Tris-HCl pH8.5, 8.66mM MgCl 2 , 70mM KCl, 11%v / vDMSO, 3.3mM DTT, 1.25mM of various dNTPs and NTPs, 0.1μM promoter primer F201111-1 in Table 5, 0.1μM reverse primer R708 in Tabl...

Embodiment 3

[0113] Example 3: Amplification of Treponema pallidum (treponema pallidum, TP) DNA (template is double-stranded DNA)

[0114] A conserved restriction nickase Nb.BtsI site is encoded in the conserved region of DNA polymerase I gene (polA) of TP DNA. On the negative strand, the restriction site is 3`-CGTCACˇNN-5`. TP DNA in its natural state is double-stranded, and 6 μL of TP DNA solution is used for each determination. Restriction enzyme digestion was carried out under the following conditions: TMA buffer (50mM Tris-HCl pH8.5, 8.66mM MgCl 2 , 70 mM KCl, 11% v / v DMSO, 3.3 mM DTT, 1.25 mM of various dNTPs and NTPs, 0.1 μM promoter primer F400806 in Table 7, 0.1 μM reverse primer R400806-3 in Table 7, 0.067 μM Molecular beacon probe MBP3 and 2U restriction nickase Nb.BtsI in Table 7 (experimental results show that there is no significant difference in the amplification effect of the nickase in the range of 1U-5U, and the effect of 2U is slightly better) . After incubating at 41...

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Abstract

The invention belongs to the technical field of biology, and discloses a nucleic acid amplification method and a kit thereof. The method is characterized in that restriction enzyme is additionally utilized for amplifying double-chain DNA (Desoxvribose Nucleic Acid) and / or single-chain DNA, and other reagents are not added, so that the influence of the substances on the follow-up TMA (Thyroid Microsomal Antibody) or NASBA (Nucleic Acid Sequence Based Amplification) reaction is avoided, and as a result, the problems of reduction of amplification efficiency and increase of nonspecific products caused by additionally added restrictive oligonucleotides can be avoided. The method and the kit thereof are applied to DNA of any type, virus DNA to be detected in a sample, and genome DNA, and can be used for multiple detection of a DNA mixed sample as well as multiple detection of DNA and RNA (Ribose Nucleic Acid) mixed samples. The method can be widely used in match with other relevant technologies in the fields of molecular diagnosis, scientific research, epidemic prevention detection, medicolegal expertise and other fields.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a nucleic acid amplification method. Background technique [0002] Polymerase Chain Reaction (PCR) technology is a revolutionary breakthrough in the field of molecular biology in the 1980s. After decades of improvement, PCR methods have developed from qualitative to quantitative, capable of amplifying from a few copies or a single cell to billions of specific nucleic acid fragments within a few hours. [0003] Although PCR technology has occupied the monopoly position of nucleic acid amplification technology for a long time, a series of isothermal nucleic acid amplification technologies developed subsequently are gradually becoming an alternative to PCR technology and are widely used. These amplification techniques apply different principles and methods, and can achieve nucleic acid (DNA or RNA) amplification under a specific temperature condition (such as 37°C). International and ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12Q1/6865C12Q2521/301C12Q2521/327C12Q2565/1015
Inventor 周裕程杨文秀万强
Owner SNOVA BIOTECH
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