Oligonucleotide primer, reagent kit and application

A technology of oligonucleotides and nucleotides, applied in oligonucleotide primers, kits and application fields, can solve the problems of large detection sensitivity deviation, poor uniformity, and low specificity of Ω primers, and achieve simplified operation process, Reduce time and cost, and solve the effect of non-specific bottlenecks

Pending Publication Date: 2020-08-14
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the fact that multiplex PCR in the prior art has amplification bias, amplification mutation and other factors that lead to uneven amplification of the target region, which has a biased impact on the subsequent sequencing data, and the Ω primers in the prior art have low specificity, poor uniformity and poor detect

Method used

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  • Oligonucleotide primer, reagent kit and application
  • Oligonucleotide primer, reagent kit and application
  • Oligonucleotide primer, reagent kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Construction of 24SNP Amplicon Sequencing Library for Chemotherapy

[0084] Utilize the present invention to carry out the construction of chemotherapy 24SNP amplicon sequencing library, the steps are as follows:

[0085] 1. Nucleic acid extraction

[0086] Human DNA was used as a template to amplify 24 target sequences using 24 pairs of specific primers and 1 pair of universal primers.

[0087] 2. Construction of multiplex PCR amplicon sequencing library

[0088] 2.1. Primer design and synthesis

[0089]The Primer Premier 5 primer design software was used to detect the targets related to the efficacy of chemotherapy targeting drugs (ERCC1, XRCC1, MTHFR, UGT1A1, CYP19A1, CYP2C9, NQO1, CYP1B1C, MDR1, CDA, DPYD, GSTP1, TYMS, CYP2C8, CBR3, CASP7). Primer design, a total of 24 pairs of specific primers (ie, amplicon-specific forward primer and amplicon-specific reverse primer) and 1 pair of universal primers (ie, forward universal primer and reverse universal primer). T...

Embodiment 2

[0109] Construction of human EGFR / BRAF / PIK3CA / KRAS / NRAS / HER2 / MAP2K1 amplicon sequencing library

[0110] Human DNA was used as a template, and 15 pairs of specific primers (designed according to the Omega-like primer design characteristics of the present invention) and a pair of universal primers were used for PCR amplification. 15 pairs of specific primers were used to amplify 15 mutation sites of EGFR / BRAF / PIK3CA / KRAS / NRAS / HER2 / MAP2K1 gene respectively.

[0111] The PCR reaction system (25 μL) is shown in Table 2, and two tubes of reaction solutions were prepared at the same time:

[0112] Table 2. PCR reaction system

[0113]

[0114] Tube 1 uses the PCR amplification reaction program as follows: activation at 95°C for 5min; denaturation at 95°C for 15s, annealing of specific primers at 55°C for 30s, extension at 72°C for 30s, 10 cycles. The second-stage library enrichment program was as follows: denaturation at 95°C for 15s, universal primer annealing at 60°C for 30s,...

Embodiment 3

[0119] Design 15 pairs of specific Ω primers according to the structural characteristics of the disclosed Ω primers, the amplified target sequence, the DNA template used, the PCR reaction system and the amplification reaction program (tube 2) are consistent with those in Example 2, and the other steps are all Consistent, compare the experimental effect of the Ω primer and the Omega-like primer of the present invention.

[0120] The analysis and comparison results are as follows:

[0121] (1) PCR amplification products were subjected to 1% agarose gel electrophoresis (attached Figure 6 );

[0122] (2) The read number distribution of each amplicon obtained by sequencing is shown in Table 3:

[0123] Table 3. Read distribution table of amplicons

[0124]

[0125] (3) The mutation status of each amplicon site detected by sequencing is shown in Table 4:

[0126] Table 4. Amplicon site mutation table

[0127]

[0128]

[0129] The results show that the Omega-like prim...

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Abstract

The invention belongs to the fields of a molecular biology detection technique and accurate medicines, and particularly relates to an oligonucleotide primer, a reagent kit and an application. The oligonucleotide primer comprises 3p arms at 3 'terminal and 5' terminal, a hairpin loop and 5p arms at the 3'terminal and the 5' terminal, wherein the 5p arms are not combined with a DNA template, the 3parms are hybridized with the DNA template, and besides, sequence specificity that polymerase extends is provided. The hairpin loop is located between the 5p arms and the 3p arms, and comprises a single-stranded loop hybridized with the DNA template and dual-stranded stems; and when the oligonucleotide primer is combined with the DNA template, binding energy between the single-stranded loop and theDNA template is greater than that of the dual-stranded stems. The invention further provides the reagent kit and an application thereof to multiplex PCR. The problems that a conventional primer is low in specificity, poor in homogeneity and high in detection sensitivity deviation, multiplex PCR primer dimers are formed, amplification preference is high, and mispairing rate is high, are solved, and the multiplex PCR effect is stable and reliable.

Description

technical field [0001] The invention belongs to the field of molecular biology detection technology and precision medicine, and specifically relates to an oligonucleotide primer, a kit and an application. Background technique [0002] With the development of high-throughput sequencing technology and the reduction of sequencing costs, its research and application in the fields of life sciences and medicine are becoming more and more extensive, especially in the diagnosis and prevention of diseases. Screening, tumor diagnosis, major disease prevention, health-related metagenomic analysis, etc. Although human whole genome sequencing has made a huge leap forward in terms of sequencing time and capital cost, the huge data analysis and genetic information extraction are time-consuming and laborious. High-coverage sequencing can also detect low-frequency mutations. Using amplicon sequencing, researchers can focus on key regions of interest in the genome. This highly targeted app...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q2531/113C12Q2537/143C12Q2525/301
Inventor 刘文干蒋艳鑫韦玉军
Owner 安徽安龙基因科技有限公司
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