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CN110603328A - Quantitative PCR amplification primer pair and application thereof - Google Patents

Quantitative PCR amplification primer pair and application thereof Download PDF

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CN110603328A
CN110603328A CN201780090496.8A CN201780090496A CN110603328A CN 110603328 A CN110603328 A CN 110603328A CN 201780090496 A CN201780090496 A CN 201780090496A CN 110603328 A CN110603328 A CN 110603328A
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primer
pcr amplification
quantitative pcr
sequence
primers
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CN110603328B (en
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杨林
高雅
张艳艳
张海萍
黄国栋
陈芳
徐惠欣
蒋慧
徐讯
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BGI Shenzhen Co Ltd
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

提供了定量PCR扩增引物对，其包括：第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，并且，第一特异性序列和第二特异性序列分别为针对靶序列的上游引物和下游引物，第一随机序列和第二随机序列反向互补，所述第一引物的5’末端碱基和所述第二引物的5’末端碱基均连接有荧光基团，且距离所述第一引物的5’末端碱基和所述第二引物的5’末端碱基特定长度的碱基均连接有淬灭基团。A quantitative PCR amplification primer pair is provided, comprising: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is located in the The 3' end of a primer, the first random sequence is located at the 5' end of the first primer, the second primer contains a second specific sequence and a second random sequence, the second specific sequence is located at the 5' end of the first primer. The 3' end of the second primer, the second random sequence is located at the 5' end of the second primer, and the first specific sequence and the second specific sequence are respectively the upstream primer and the downstream primer for the target sequence , the first random sequence and the second random sequence are reverse complementary, the 5' end base of the first primer and the 5' end base of the second primer are both connected with a fluorophore, and the distance from the first primer The 5' terminal base of the primer and the base of a specific length of the 5' terminal base of the second primer are connected with a quenching group.

Description

PCT国内申请，说明书已公开。PCT domestic application, the description has been published.

Claims (13)

  1. A quantitative PCR amplification primer pair, comprising:
    a first primer and a second primer, wherein the first primer and the second primer are different,
    wherein,
    the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is positioned at the 3 'end of the first primer, and the first random sequence is positioned at the 5' end of the first primer,
    the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is positioned at the 3 'end of the second primer, and the second random sequence is positioned at the 5' end of the second primer,
    and,
    the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at a target sequence, and the first random sequence and the second random sequence are reversely complementary,
    the 5 'end base of the first primer and the 5' end base of the second primer are both connected with a fluorescent group, and the bases which are away from the 5 'end base of the first primer and the 5' end base of the second primer by a certain length are both connected with a quenching group.
  2. The pair of primers for quantitative PCR amplification according to claim 1, wherein the first and second specific sequences have a TM value of 55-65 ℃ and the first and second primers have a TM value of 65-75 ℃.
  3. The pair of primers for quantitative PCR amplification according to claim 1, wherein the first random sequence and the second random sequence have a length of 16-30bp, and the first specific sequence and the second specific sequence have a length of 16-30 bp.
  4. The pair of primers for quantitative PCR amplification according to claim 1, wherein the 2 nd to 5 th bases at the 5 'ends of the first and second primers are modified by thio, and the 1 st to 5 th bases at the 3' ends of the first and second primers are modified by thio.
  5. The pair of primers for quantitative PCR amplification according to claim 4, wherein the thio-modification is any one selected from the group consisting of a phosphorothioate-type modification, a methylsulfate-type modification and a peptide nucleic acid modification.
  6. The pair of primers for quantitative PCR amplification according to claim 1, wherein at least one of the first primer and the second primer has a 5' end modified by phosphorylation.
  7. The pair of primers for quantitative PCR amplification according to claim 1, wherein the quencher group is linked to a base 10 to 30bp away from the 5 'terminal base of the first primer and the 5' terminal base of the second primer.
  8. A quantitative PCR amplification kit comprising the pair of quantitative PCR amplification primers according to any one of claims 1 to 7.
  9. A quantitative PCR amplification method, wherein the quantitative PCR amplification is performed using the quantitative PCR amplification primer set according to any one of claims 1 to 7 or the quantitative PCR amplification kit according to claim 8.
  10. The method of claim 9, wherein the method comprises two rounds of amplification comprising:
    carrying out first round linear amplification on the quantitative PCR amplification primer pair and the template at an annealing temperature of 55-65 ℃; and
    and carrying out second round of circular amplification on the products of the first round of linear amplification at the annealing temperature of 65-72 ℃.
  11. The method of claim 10, wherein the amplification reaction sequence of the method is as follows:
    Figure PCTCN2017089197-APPB-100001
  12. a method for quantitative analysis of a DNA sample to be tested, comprising:
    the method according to any one of claims 9 to 11, wherein the DNA sample to be tested is subjected to fluorescent quantitative PCR amplification and the quantitative analysis is carried out based on the collected fluorescent signal.
  13. A method for performing gene expression differential analysis of a specific gene in a plurality of test DNA samples, wherein each of the plurality of test DNA samples comprises a cDNA sequence of the specific gene, the method comprising:
    the method according to any one of claims 9 to 11, wherein the plurality of DNA samples to be tested are subjected to fluorescent quantitative PCR amplification respectively, and quantitative analysis is realized based on the collected fluorescent signals; and
    comparing the quantitative analysis results of the plurality of DNA samples to be tested so as to determine the gene expression difference of the specific genes of the plurality of DNA samples to be tested.
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Cited By (1)

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CN114875116A (en) * 2022-04-27 2022-08-09 广州博懿瑞生物科技有限公司 A self-quenching fluorescence primer and its design method and application

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GB2512631A (en) * 2013-04-03 2014-10-08 Rupert Maxwell Gaut Quantitative detection of specific nucleic acid sequences
CN104404142A (en) * 2014-11-11 2015-03-11 中国科学院上海微系统与信息技术研究所 Fluorescent probe for fluorescent quantitative PCR reactions
US20160237472A1 (en) * 2012-11-30 2016-08-18 Beijing Tag-Array Molecular Test Co., Ltd Primer middle sequence interference pcr technology
US20160369327A1 (en) * 2013-06-18 2016-12-22 Lgc Limited Fluorophore-based oligonucleotide probes with a universal element

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US20160237472A1 (en) * 2012-11-30 2016-08-18 Beijing Tag-Array Molecular Test Co., Ltd Primer middle sequence interference pcr technology
GB2512631A (en) * 2013-04-03 2014-10-08 Rupert Maxwell Gaut Quantitative detection of specific nucleic acid sequences
US20160369327A1 (en) * 2013-06-18 2016-12-22 Lgc Limited Fluorophore-based oligonucleotide probes with a universal element
CN104404142A (en) * 2014-11-11 2015-03-11 中国科学院上海微系统与信息技术研究所 Fluorescent probe for fluorescent quantitative PCR reactions

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JURATE BITINAITE等: "USER™ friendly DNA engineering and cloning method by uracil excision", NUCLEIC ACIDS RES, vol. 35, no. 6, pages 1992 - 2002, XP055170276, DOI: 10.1093/nar/gkm041 *
郭秋平等: "用荧光双链引物特异扩增并定量核酸", 《厦门大学学报(自然科学版)》 *
郭秋平等: "用荧光双链引物特异扩增并定量核酸", 《厦门大学学报(自然科学版)》, vol. 41, no. 01, 10 February 2002 (2002-02-10), pages 108 - 111 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114875116A (en) * 2022-04-27 2022-08-09 广州博懿瑞生物科技有限公司 A self-quenching fluorescence primer and its design method and application
CN114875116B (en) * 2022-04-27 2023-08-29 广州博懿瑞生物科技有限公司 Self-quenching fluorescence primer and design method and application thereof

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