Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample

A DNA library and fragmentation technology, applied in the biological field, can solve problems such as difficulty in building a library of trace FFPE samples

Active Publication Date: 2015-09-30
天津诺禾医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The present invention aims to provide a method for fragmenting a trace DNA sample and a method for constructing a DNA library...

Method used

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  • Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample
  • Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample
  • Method for fragmenting trace DNA sample and method for establishing DNA library by utilizing trace DNA sample

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Experimental program
Comparison scheme
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Embodiment 1

[0055] 2 cases of FFPE samples from patients with non-small cell lung cancer, the sample names and extraction volumes are shown in Table 2.

[0056] Table 2

[0057]

[0058] 1) Fragmentation of DNA

[0059] Use an ultrasonic breaker (Covaris LE220R) to fragment the target DNA and the incorporated Carrier RNA (500ng) according to specific shearing parameters. The shearing parameter settings of Covaris LE220R are shown in Table 1.

[0060] The fragmented DNA was purified by 1.8× AMPure XP magnetic beads before proceeding to the next step.

[0061] 2) End repair and A base addition at the 3' end

[0062] Refer to the ratio in Table 3 below to prepare the reaction mixture, and gently blow up and down with a gun to mix well.

[0063] table 3

[0064]

[0065] Put it into the PCR machine, and set the program according to the table below for reaction (set the cover temperature to 70°C, do not cover the heated cover for the first 30 minutes, and immediately cover the heated...

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Abstract

The invention discloses a method for fragmenting a trace DNA sample and a method for establishing a DNA library by utilizing the trace DNA sample. The method for fragmenting the trace DNA sample comprises the steps that 1, Carrier RNA is added into the trace DNA sample to obtain a sample to be fragmented; 2, fragmenting processing is conducted on the sample to be fragmented. An inventor discovers that after the exogenetic Carrier RNA is added into the target DNA, that is to say, the target DNA is mixed with the Carrier RNA and then fragmenting is conducted on the mixture, the protective action to the target DNA can be achieved, the destructive action of a fragmenting instrument to the trace DNA is reduced to the maximum extent, and full gene establishing library sequencing and even hybrid capture sequencing can be conducted on DNA of a trace FFPE sample.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for fragmenting a trace DNA sample and a method for constructing a DNA library using a trace DNA sample. Background technique [0002] Formalin-fixed paraffin-embedded (FFPE) is an important method of preserving tissue samples, which is widely used in clinical and scientific research fields, including morphological and pathological research and molecular level Research. However, it is very challenging to study FFPE samples at the molecular level. There are three main reasons. First, FFPE medical samples are very precious, and the content of each sample is limited, so many samples cannot be obtained again later. Lin began to degrade before soaking, and the cross-linking produced during formalin penetration made the extraction work more difficult. In addition, most FFPE samples were stored for a long time, and the storage environment and time had a great impact on the DNA in ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C40B50/06
Inventor 蒋智张振宇曹志生杜长诗魏勤
Owner 天津诺禾医学检验所有限公司
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