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Base balance-based molecular marker method for amplicon

A technology of molecular labeling and base balance, which is applied in the field of molecular biology and can solve problems such as base imbalance

Active Publication Date: 2020-06-05
北京博奥汇玖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiencies of the prior art, the present invention provides a base-balanced amplicon molecular labeling method, which solves the problem of severe base imbalance in amplicon sequencing while effectively distinguishing samples

Method used

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  • Base balance-based molecular marker method for amplicon
  • Base balance-based molecular marker method for amplicon
  • Base balance-based molecular marker method for amplicon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0030] 341F forward primer containing molecular markers:

[0031]

[0032]

[0033] 805R reverse primer containing molecular markers:

[0034] name sequence(5'to3') 805R-1 GCNGCATNNNAHGACTACHVGGGTATCTAATCC 805R-2 TANTAGCNNNGHGACTACHVGGGTATCTAATCC 805R-3 CGNCGTANNNTHGACTACHVGGGTATCTAATCC 805R-4 ATNATCGNNNCHGACTACHVGGGTATCTAATCC 805R-5 GANAGTCNNNNCHGACTACHVGGGTATCTAATCC 805R-6 TGNGTCANNNNAHGACTACHVGGGTATCTAATCC 805R-7 CTNTCAGNNNGHGACTACHVGGGTATCTAATCC 805R-8 ACNCAGTNNNTHGACTACHVGGGTATCTAATCC 805R-9 CGNAACTNNNNGHGACTACHVGGGTATCTAATCC 805R-10 ATNGGANNNNTHGACTACHVGGGTATCTAATCC 805R-11 GCNTTGANNNCHGACTACHVGGGTATCTAATCC 805R-12 TANCCTGNNNAHGACTACHVGGGTATCTAATCC

[0035] 519F forward primer containing molecular markers:

[0036]

[0037]

[0038] 926R reverse primer containing molecular markers:

[0039] name sequence(5'to3') 926R-1 GCNGCATNNNADCCGTCAATTCMTTTRAGTTTT 9...

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Abstract

The invention discloses a base balance-based molecular marker method for an amplicon, and relates to the technical field of molecular biology. A 5' end of a forward primer and a 5' end of a reverse primer of the amplicon each have a molecular marker of a12-base; in each molecular marker, bases at 4 positions are degenerate bases N, namely that, the bases at the 4 positions can be any one of A / C / G / T four bases; bases at 7 positions are definite single base, A / C / G / T; and a base at a last position is a non-N degenerate base. According to the base balance-based molecular marker method for the amplicon, the bases at the last positions of the molecular markers of the forward primer and the reverse primer are degenerate bases consisting of all possible bases after corresponding complementary bases at the positions in conservative regions, namely that, bases and complementary bases of conventional primers that should appear at the positions are excluded and in such a manner, the base imbalanceproblem in amplicon sequencing can be greatly alleviated while samples are effectively distinguished, and the melting temperature of the primers to which the molecular markers are added is closer totheoretical expectations.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a base-balanced amplicon molecular labeling method. It is suitable for amplicon sequencing of genes such as 16S / 18S / ITS or intergenic regions. Through the base balance strategy, it can greatly alleviate the problem of base imbalance in amplicon sequencing while effectively distinguishing samples. Background technique [0002] When analyzing the microbial community structure, the commonly used molecular biology method is to select a gene segment that can distinguish species (such as the 16S gene region of bacteria or the 18S / ITS gene region of fungi) to amplify, and then sequence the The base sequence of the amplified region is read, and then the read sequence is compared with the known species sequence in the database for similarity comparison, and finally the corresponding species count is obtained. [0003] When doing high-throughput sequencing, since the sequence is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122
Inventor 刘彬
Owner 北京博奥汇玖生物科技有限公司
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