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Novel amplification method and application of ligase reaction mediate

A ligase reaction, mediated technology, applied in the field of amplification, can solve the problems of false positives and limited sensitivity, and achieve the effects of high specificity, efficient amplification and detection, easy design and chemical synthesis

Active Publication Date: 2014-06-04
爱基因鑫享(厦门)医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LDR / PCR and MLPA technologies are affected by these two non-specific effects, so the detection sensitivity is limited, and non-specific signals can be detected, and there is a risk of false positives
PLP and MIP technologies use the method of exonuclease to digest unconnected hybridization probes, which eliminates the non-specific amplification of hybridization probes in the PCR reaction to a certain extent, but when the detection multiplicity is high, it will still be affected by non-specific amplification. The influence of specific connection, the appearance of false positive is still inevitable

Method used

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  • Novel amplification method and application of ligase reaction mediate
  • Novel amplification method and application of ligase reaction mediate
  • Novel amplification method and application of ligase reaction mediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Comparison of melting curves of multiple connection probes:

[0033] target sequence: CTGCAGGGAAATG TCTAGATTGGATCTTGC (SEQ ID NO: 1)

[0034] Ligation probe 1 (hybridizes fully complementary to the target sequence):

[0035] GCAAGATCCAATCTAGACATTTCCCTGCAG (SEQ ID NO: 2)

[0036] Ligation probe 2 (hybridizes with the target sequence to form a hybrid community with a capsule structure, the capsule structure is located in the middle, and the bases at the dotted line form the capsule structure):

[0037] GCAAGATCCAATCTA GGAATCTGGATTCAAAATCTTGACATTTCCCTGCAG

[0038] (SEQ ID NO:3)

[0039] Ligation probe 3 (hybridizes with the target sequence to form a hybrid community of a capsule structure, and the bases at the dotted line position form a capsule structure):

[0040] GCAAGATCCAATCTAGACATTT GGAATCTGGATTCAAAATCTTCCCTGCAG

[0041] (SEQ ID NO:4)

[0042] Ligation probe 4 (hybridizes with the target sequence to form a hybrid community of a capsule struc...

Embodiment 2

[0049] Embodiment 2: Quantitative detection target sequence

[0050] Select an artificially synthesized DNA sequence as the object of investigation, and design three ligation probes for this sequence to hybridize to the positions adjacent to the target sequence. The specific sequence is as follows:

[0051] target sequence:

[0052] CTACACAGTCTCCTGTACCTGGGCAATATGATGCTACCAAATTTAAGCAGTATAGCAGACATGTTGAGGAATATGA (SEQ ID NO: 7)

[0053] Connect probe a:

[0054] TGGAGCGACGATACGAAGATA TCATATTCCTCAACATGTCTGC (SEQ ID NO: 8)

[0055] Connect probe b:

[0056] PO4-TATACTGCTTAAATTT AACTTCGGTCCTTCATCGCT GGTAGCATCATATTGC

[0057] (SEQ ID NO:9)

[0058] Connect probe c:

[0059] PO4-CCAGGTACAGGAGACTGTGTAG TCTAGATAGGATCTTGGAGC

[0060] (SEQ ID NO: 10)

[0061] Wherein, the upstream primer tag sequence of connection probe a is the universal primer F sequence, and the downstream primer binding tag sequence of connectio...

Embodiment 3

[0068] Example 3: SNP Genotyping Detection of Three Human Genomic DNA Samples

[0069] Select the SNP site (rs740598) as the research object, and design four connection probes as follows:

[0070] Connect probe a:

[0071] TGGAGCGACGATACGAAGATA CCAAATATTTTTCGTAAGTATTTCAAAT

[0072] (SEQ ID NO: 14)

[0073]To connect probe b-1:

[0074] PO4-AGCAATGGCTCGTC CATCTCTAAGGCAAGGCTC TATGGTTAGTCTCA

[0075] (SEQ ID NO: 15)

[0076] To connect probe b-2:

[0077] PO4-AGCAATGGCTCGTC ACCTTCCGTCTGTACTCGT TATGGTTAGTCTCG

[0078] (SEQ ID NO: 16)

[0079] Connect probe c:

[0080] PO4-CAGCCACATTCTCAGAACTGC TCTAGATAGGATCTTGGAGC (SEQ ID NO: 17)

[0081] Wherein, the tag sequences of connecting probes a and c are the same as in Example 2, and the detection tag sequence of connecting probe b-1 is a FAM-labeled Taqman probe sequence (FAM-CATCTCTAAGGCAAGGCTC...

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Abstract

Disclosed are a novel ligase reaction mediated amplification method and use thereof. General amplification and detection downstream are implemented through the ligase reaction of three ligation probes. Further disclosed are a kit and chip containing three ligation probes and uses thereof, and especially the use in genotyping detection of gene sequences.

Description

technical field [0001] The invention relates to an amplification method, in particular to a ligase reaction-mediated amplification method. Background technique [0002] Nucleic acid detection technology has been widely used in clinical diagnosis in molecular genetics, immunology, oncology and microbiology. With the rapid development of molecular biology, some new nucleic acid detection methods are also emerging, among which, ligase-dependent detection technology is one of them. Ligase is an enzyme that closes the gap (Nick) on the DNA or RNA chain, with the help of NAD + Or the energy provided by the hydrolysis of ATP catalyzes the reaction of the 3' terminal hydroxyl group and the 5' terminal phosphate group of two nucleic acid single strands to form a phosphodiester bond. Ligase-dependent detection techniques are primarily enabled by the principle of nucleic acid hybridization and its fidelity at nick junctions. In 1988, Landegren first invented the ligase in vitro muta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12Q1/68C12Q1/6862C12Q1/6848
Inventor 王小波
Owner 爱基因鑫享(厦门)医学检验实验室有限公司
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