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Library preparation method of trace nucleic acid sample and application thereof

一种核酸文库、核酸样本的技术，应用在微量核酸样本的文库制备领域，能够解决阻碍单细胞和微量核酸应用等问题

Active Publication Date: 2013-04-24
BGI BIOTECH WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the preparation of the library requires the amount of initial DNA / RNA in micrograms. Although the initial amount of library construction can be reduced after optimization, it is still impossible to directly prepare the library for single-cell or extremely small nucleic acid samples, so it is seriously Hinders the application of single-cell and micro-nucleic acid sequencing

Method used

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  • Library preparation method of trace nucleic acid sample and application thereof
  • Library preparation method of trace nucleic acid sample and application thereof
  • Library preparation method of trace nucleic acid sample and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Preparation of single-cell genomic DNA library by DOP-PCR method

[0105] In this embodiment, a single lymphocyte in the blood is taken as an example, and a single-cell genomic DNA library is prepared by the DOP-PCR method.

[0106] 1. Sample source:

[0107] The blood single cell samples came from the peripheral blood of a normal person (YH, Yanhuang Project sample) and a patient with Down syndrome (T21).

[0108] 2. Single cell separation:

[0109] A total of 3 YH and 1 T21 peripheral blood single lymphocytes were separated by mouth pipette method and placed in a PCR tube containing 2 μl alkaline cell lysate (200 mM KOH, 50 mM DTT) and stored at -80° C. for more than 30 minutes.

[0110] 3. The first PCR (low stringency amplification, DOP pre-amplification):

[0111] DOP pre-amplification The PCR tube containing single cells was treated at 65°C for 15 minutes, and then DOP reaction solution was added to perform DOP pre-amplification reaction. In this example, pfx DNA polymerase ...

Embodiment 2

[0168] Repeat Example 1, the difference lies in the DOP pre-amplification reaction conditions, samples and other experimental conditions are the same. The DOP pre-amplification reaction conditions are shown in Table 8.

[0169] Table 8

[0170]

[0171] The results show that the DOP pre-amplification reaction conditions of this example can also achieve the purpose of constructing a library and using it for the next step of detection.

Embodiment 3

[0173] DOP-PCR method for preparing library of trace DNA / cDNA samples

[0174] The sample in this embodiment is a trace DNA / cDNA sample, including trace genomic DNA, immunoprecipitation (IP) product DNA, plasma free DNA (Plasma DNA), and RNA reverse transcription cDNA product.

[0175] The samples of IP product DNA, plasma free DNA (Plasma DNA), and genomic DNA (gDNA) were all diluted 5-fold, and the library was prepared starting with 200pg, 40pg, and 8pg respectively.

[0176] The cDNA sample was obtained by reverse transcription of 1μg of total mouse RNA with hexanucleotide random primers by Superscript II reverse transcriptase, and was diluted 5-fold to the original concentration and 5 -1 , 5 -2 , 5 -3 The concentration starts with library preparation.

[0177] A small amount of DNA / cDNA sample is directly added to the reaction solution to perform the DOP pre-amplification reaction. This embodiment still uses pfx polymerase as an example.

[0178] The reaction system was prepared acc...

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Abstract

The invention relates to a library preparation method of a trace nucleic acid sample and application thereof. The method comprises: using a DOP (Degenerate Oligonucleotide Primed) primer to perform first amplification on DNA in the sample, with a DOP primer sequence having at least a 5' terminal nondegenerate nucleotide zone and a 3' terminal degenerate nucleotide zone; employing a DOP-Amp primer to conduct second amplification on a first PCR product so as to obtain a second PCR amplification product; and carrying out adaptor-ligation PCR on the second PCR amplification product to obtain a third PCR product, and taking the third PCR product with sequencing adaptors on both ends as the nucleic acid library. The library can be used for fragment size selection and high-throughput sequencing to obtain gene information related to diseases in the sample. The invention also provides a kit and its application in preparation of the trace nucleic acid sample library.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for preparing a library of trace nucleic acid samples and its application. Background technique [0002] Next-generation sequencing technology (NGS) has changed the previous analysis mode of DNA / RNA samples and has become an indispensable research tool in almost all research fields. The next-generation sequencing technology uses parallel sequencing of millions of short DNA fragments at the same time, so that the sequencing of each base can be completed in a short time, and the cost is greatly reduced. NGS technology has been applied in many aspects, such as genomics, transcriptomics, epigenomics, clinical diagnosis, etc. [0003] There are several next-generation sequencing technology NGS platforms on the market, including Genome Analyzer, Hiseq, and Miseq series sequencing platforms from Illumina, 454 sequencing platforms from Roche, SOLID sequencing pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06C12N15/10C12Q1/68
CPCC40B50/06C12Q1/6874C12N15/10C12Q1/686C40B40/06C12N15/1093C12Q1/6853C12Q1/6869C12Q2523/125C12Q2525/179C12Q2525/191C12Q2531/113C12Q2537/159
Inventor 殷旭阳张春雷蒋慧张秀清
Owner BGI BIOTECH WUHAN CO LTD
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