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Novel DNA library construction method and application thereof

Another sticky-end technology, applied in the field of molecular biology, can solve the problems of loss of DNA information, inability to use DNA information effectively, and poor linking efficiency of adapters.

Inactive Publication Date: 2018-06-15
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the PCR amplification method is biased towards fragments, and generally can only cover areas with better quality, and the amplification situation of samples with breakpoints is very unsatisfactory, and all DNA information in the samples cannot be effectively used; while in double-stranded In the ligation scheme, the efficiency of adapter ligation is generally poor in regions that are greatly affected by library fragmentation, which will also lead to the loss of DNA information

Method used

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  • Novel DNA library construction method and application thereof
  • Novel DNA library construction method and application thereof
  • Novel DNA library construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202] 1. Test preparation

[0203] (1) Synthesis of cohesive end adapters:

[0204] The sticky end adapter is a double-stranded linker with a sticky end, one end of the sticky end adapter is a first sticky end, and the first sticky end includes at least a random sequence.

[0205] 1. As an example, use Primer1 and Primer2 to synthesize sticky end adapters,

[0206] Primer1: 5'-AGATCGGAAG-3'-bioten (SEQ ID NO.1);

[0207] Primer2: 5'-CTTCCGATCTNNNNNN-3' (SEQ ID NO.2); wherein, NNNNNN represents a random sequence, usually the length of the random sequence is 2-30nt, preferably 4-20nt, more preferably 5-10nt .

[0208] 2. Add the configuration reaction solution according to Table 1:

[0209] Table 1

[0210] Reagent

Volume (ul)

Primer1 (10μM)

1

Primer2 (10μM)

1

TE buffer

7

5M NaCl

1

total capacity

10

[0211] The TE buffer used in the reaction can also be replaced by water, Tris-Hcl (0.05mM, pH7.0-8.0) and...

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Abstract

The invention relates to the technical field of molecular biology and in particular relates to a novel DNA library construction method and an application thereof. According to the DNA library construction method disclosed by the invention, an adhesive end joint can be directly mediated by a common ligase to be connected to a single-stranded DNA denatured from a sample genome DNA; the novel DNA library construction method can be applied to a sample with low initial mass; and compared with previous methods, the novel DNA library construction method has the advantages that the connection efficiency of samples is greatly increased, the diversity of the samples are retained to the greater extent, the stability is good, and library bias caused by artificial factors is shortened.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a novel DNA library construction method and its application. Background technique [0002] The function of DNA molecules is to store all the genetic information of almost all proteins and RNA molecules that determine the traits of species. The interpretation of DNA information is of great significance for us to study the mysteries of life and the mechanism of diseases. DNA molecules usually exist in samples in the form of double strands, but the vast majority of precious medical samples exist in the form of formalin-fixed paraffin-embedded (FFPE) samples. [0003] FFPE samples are precious, but there are difficulties in amplification in molecular biology experiments. Usually FFPE is of poor quality, fragmented and has many breakpoints on the DNA, which poses certain difficulties for the amplification of the corresponding samples, because usually the polymerase cannot s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/10C12Q1/6869
CPCC12N15/1093C12Q1/6869C40B50/06C12Q2525/191C12Q2521/501C12Q2533/101
Inventor 李静陈昌岳王芳王雨倩赵慧茹胡秋萍张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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