Method for enriching DNA target regions by synergistic PCR method based on artificial nucleic acid analogue technology

An enrichment and nucleotide technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as uneven amplification products, difficulty in primer design, and difficulty in optimizing amplification conditions

Pending Publication Date: 2020-07-17
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] PCR technology is a method for quickly obtaining specific DNA sequences through in vitro enzymatic amplification reactions pioneered by Mullis in 1983. It has been widely used in molecular biology, molecular genetics, medicine and other fields, but traditional PCR has complicated amplification Sometimes it still doesn't work well when templating
For example, for templates with high GC content, due to non-specific interactions, it is often difficult to optimize suitable amplification conditions; for another example, when amplifying less than 100 copies of template DNA, two problems will be encountered: one is the formation of primers Dimer, and the second is that non-specific amplifica

Method used

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  • Method for enriching DNA target regions by synergistic PCR method based on artificial nucleic acid analogue technology
  • Method for enriching DNA target regions by synergistic PCR method based on artificial nucleic acid analogue technology
  • Method for enriching DNA target regions by synergistic PCR method based on artificial nucleic acid analogue technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] The design of embodiment 1, ANABB-PCR primer

[0080] In this embodiment, the partial sequence of the human genome TP53 gene (this partial sequence is shown in sequence 1 of the sequence listing, 2530bp) is selected as an example, the primer is designed, and the specific effect of the primer is verified in Example 2 and Example 3 / implementation In Example 4, the effect of primers on the amplification of complex templates was verified.

[0081] The natural base primer set consists of a natural base inner primer pair and a natural base outer primer pair. The primer pair inside the natural base is composed of primer F1 (upstream primer inside the natural base) and primer R1 (downstream primer inside the natural base). The primer pair outside the natural base is composed of primer F2 (upstream primer outside the natural base) and primer R2 (downstream primer outside the natural base).

[0082] The ANABB primer set consists of ANABB inner primer pair and ANABB outer primer...

Embodiment 2

[0083] Embodiment 2, the specificity of non-natural base primer

[0084] Primers to be tested: primer 1, primer 2 (introduced 2 Ds), primer 3 (introduced 3 Ds), primer 4 (introduced 4 Ds).

[0085] 1. Comparison of primer self-dimer effects

[0086] Carry out the following test for each primer to be tested respectively.

[0087] Reaction system (pH7.0, 20μl): from 10μl 480SYBR Green I Master, 1 μl primer solution to be tested and ddH 2 O composition. In the reaction system, the concentration of the primer to be tested is 0.2 μM.

[0088] After the reaction system was prepared, real-time fluorescence quantitative PCR was performed.

[0089] Reaction program: pre-denaturation at 95°C for 10 min; 40 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 20 s, and extension at 72°C for 60 s; fluorescence signals were collected at 72°C.

[0090] see results Figure 4 . Introducing non-natural nucleotides into primers can effectively avoid the formation of primer se...

Embodiment 3

[0098] Embodiment 3, the amplification effect of ANABB primer set to complex template

[0099] In this example, human genomic DNA was used as a template, and each primer set in Example 1 was used to perform PCR amplification on a 2530 bp longer template of the TP53 gene, so as to verify the effect of the ANABB primer set on complex template amplification.

[0100] 1. Amplification of ANABB primer set (test set)

[0101] The first round of PCR reaction: reaction system (pH7.0, 20 μl): by 10 μl 480SYBR GreenIMaster, 0.1 μl primer F3 solution, 0.1 μl primer R3 solution, 1 μl template solution, 0.5 μl dPxTP solution and ddH 2 O composition. In 1 μl template solution, the genomic DNA content is 100ng. In the reaction system, the concentration of primer F3 was 0.02 μM, the concentration of primer R3 was 0.02 μM, and the concentration of dPxTP was 0.5 mM. Reaction program: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 10 s, annealing at 60°C for 20 s, extensi...

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Abstract

The invention discloses a technology for enriching DNA target regions by a synergistic PCR method based on an artificial nucleic acid analogue technology. The invention protects a specific primer setcomposed of an inner primer pair and an outer primer pair; the inner upstream primer includes a section I and a section II, and the inner downstream primer includes a section III and a section IV; thesection II and the section IV are for a certain target sequence; and both the outer upstream primer and the outer downstream primer have non-natural nucleotides. The invention also protects a methodfor enriching the target sequence, and the method comprises the following steps: taking DNA containing the target sequence as a template, and performing PCR amplification by using the specific primerset, wherein the PCR amplification reaction system has paired deoxynucleotides. On the basis of synergistic PCR, the invention introduces unnatural base pair tail primer design, so that non-specific amplification and primer dimer formation can be effectively avoided, various background interferences can be greatly reduced, and the amplification effect is almost perfect. The method has great application prospects in the field of massively parallel sequencing.

Description

technical field [0001] The invention relates to a method for enriching a DNA target region by a synergistic PCR method based on artificially simulated nucleic acid technology. Background technique [0002] Artificial Nucleic Acid Analogues (Artificial Nucleic Acid Analogues) refers to a kind of artificial nucleic acid that can exercise or simulate the function of natural nucleic acid and is relatively independent by artificially designing and synthesizing through the modification or transformation of nucleotides. Those skilled in the art know that nucleotides are composed of bases and sugar ring skeletons. Therefore, the modification or transformation of nucleotides is also divided into two categories: one is the modification and transformation of sugar ring skeletons, such as Locked nucleic acid and peptide nucleic acid are representatives of this category; the other category is the modification of bases, such as early isocytosine / 5-methylisocytosine (isoC / isoMeC) and isogu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/6806
CPCC12Q1/6876C12Q1/6806C12Q2531/113
Inventor 葛猛余倩夏霞宇王宏伟
Owner 北京旌准医疗科技有限公司
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