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Method for improving PNA-based PCR inhibition efficiency, and application

A technology of inhibition efficiency and number of bases, applied in biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of small number of DNA fragments, large background DNA residue, and difficulty in detecting DNA fragments, so as to improve efficiency Effect

Pending Publication Date: 2021-01-05
浙江原创医疗科技有限公司
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  • Application Information

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Problems solved by technology

[0002] Detection of cleaved DNA fragments, circulating nucleic acids, and tumor-derived DNA is of great interest in diagnostics and the emerging field of pharmacogenetics, but generally, the number of DNA fragments is very small and there is a margin of background DNA. are much larger, therefore, there is great difficulty in detecting these types of DNA fragments

Method used

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  • Method for improving PNA-based PCR inhibition efficiency, and application
  • Method for improving PNA-based PCR inhibition efficiency, and application
  • Method for improving PNA-based PCR inhibition efficiency, and application

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Design SRY-PF1 and SRY-PR1 primer sets, as shown in Table 1, the restriction site is between AAAG and CTGTAACTCTA, that is, before the first base of the overlapping sequence. According to the usual theory, attention should be paid to the PNA of the overlapping sequence The / DNA Tm value should be 3-5°C lower than the corresponding primer / DNA Tm value.

[0018] PCR reactions were performed in the absence (-) or presence (+) of the corresponding PNAs (SRY-PNA-F and SRY-PNA-R). Electrophoresis results such as figure 1 As shown, it can be seen that PNA cannot effectively inhibit PCR amplification (swimming lanes 1&2, figure 1 , the substrates of lanes 1-6 are all adult male whole blood DNA genomic DNA extracted by the general method, without enzyme digestion).

[0019] Table 1. Sequences of PNA and PCR primers used

[0020]

[0021] Single-base mismatch primers SRY-PF1m and SRY-PR1m were designed. The mismatch site was located in the middle of the overlapping sequence...

Embodiment 2

[0026] To generate short DNA fragments, the SRY gene was digested by restriction endonuclease Alu1. Enzymatic reaction: 100 units of Alu 1 digested 1 μg of genomic DNA at 37°C for 16 hours. The enzymatic reaction was terminated by heating to 65°C for 20 minutes. After the enzymatic reaction, in the absence (-) or presence (+) of PNA, different PCR primer sets were used to amplify the digested SRY gene, and the results were as follows figure 2 shown. SRY-PF1 and SRY-PR1 are perfectly complementary to the target sequence in the template, while SRY-PF1m and SRY-PR1m have a single base mismatch with the template. figure 2 It shows that single base mismatch primers do not affect the amplification of short DNA fragments. The short DNA here refers to the short DNA after digestion.

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Abstract

The invention discloses a method for improving PNA-based PCR inhibition efficiency. The method is characterized in that a nucleic acid amplification primer adopts a single-base mismatch primer, and amismatch site is positioned in the middle of an overlapping sequence of a PNA probe and the amplification primer. According to the method, the single-base mismatch primer is used, so that the PNA-based PCR inhibition efficiency can be greatly improved. According to the method, a short DNA fragment is selectively amplified, and the PCR amplification of large background DNA is inhibited by combiningwith the single-base mismatch primer, so that the PNA can effectively detect a specific DNA fragment, such as can be applied to monitor circulating tumor DNA and circulating fetal DNA.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and more specifically relates to a method for improving the inhibition efficiency of PCR based on PNA. Background technique [0002] Detection of cleaved DNA fragments, circulating nucleic acids, and tumor-derived DNA is of great interest in diagnostics and the emerging field of pharmacogenetics, but generally, the number of DNA fragments is very small and there is a margin of background DNA. are much larger, and therefore, there are great difficulties in detecting these types of DNA fragments. [0003] Peptide nucleic acid (PNA) is a DNA analog with a polypeptide-like backbone. The main chain backbone of PNA is formed by linking N(2-aminoethyl)-glycine and nucleic acid bases through methylene carbonyl. PNA can specifically hybridize with DNA or RNA to form a stable complex. PNA has three characteristics: 1. The binding ability of PNA and DNA is better than that of DNA and DNA; 2. The stab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686
Inventor 李思慧叶盛朱晓玲徐涛梁兴国方志俊申志发小宫山真
Owner 浙江原创医疗科技有限公司
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