DNA database establishing method for high-throughput sequencing

A high-throughput, sequencing technology, applied in the field of DNA library construction for high-throughput sequencing, can solve the problems of low cfDNA content, unable to reach 10ng, and less cfDNA, and achieve low initial DNA amount and optimized reagent usage , highly specific effect

Inactive Publication Date: 2016-05-11
北京圣谷同创科技发展有限公司
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Problems solved by technology

However, when performing high-throughput sequencing on plasma cfDNA, due to the low cfDNA content in plasma, the amount of cfDNA that can be obtained is very small, especially when the amount of blood available is small, the extracted cfDNA often cannot reach 10ng. Therefore, it is necessary to Develop a method that utilizes less blood for banking

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  • DNA database establishing method for high-throughput sequencing
  • DNA database establishing method for high-throughput sequencing
  • DNA database establishing method for high-throughput sequencing

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Embodiment

[0033] Use 1ngcfDNA to build a library with the method of the present invention, which is based on the library building method of IonAmpliSeqLibraryKit2.0 (LifeTechnology), the reagents used in each step and the amount of components of the reaction system and the amount of purified magnetic beads used are halved, and for The number of PCR amplification cycles was optimized, and the specific steps are as follows:

[0034] 1) Multiplex PCR

[0035] The isolated and purified 1ng cfDNA was subjected to multiplex PCR amplification. The reaction system and conditions are shown in Table 1 and Table 2.

[0036] Table 1 Multiplex PCR reaction system

[0037] components

Add reagent volume

5X Ion AmpliSeq HiFi Master Mix

2μl

3 --> 5X Ion AmpliSeq Primer Pool

2μl

cfDNA (1ng)

xμl

nucleic acid free water

(6-x)μl

total capacity

10μl

[0038] Table 2 Multiplex PCR reaction conditions

[0039]

[0040]

[0041] ...

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Abstract

The invention provides a DNA database establishing method for high-throughput sequencing, and particularly discloses a cfDNA database establishing method. On the basis of a standard database establishing method of Ion AmpliSeq Library Kit 2.0, the agent use amount and the PCR amplification conditions are optimized, and therefore reaction consumption is reduced, amplification specificity is higher, and the initial DNA amount is lower and even lower than 1 ng.

Description

technical field [0001] The invention relates to a method for building a DNA library for high-throughput sequencing, in particular to a method for building a cfDNA library for high-throughput sequencing. Background technique [0002] Malignant tumor is one of the major diseases threatening human health, and it is also the main cause of global morbidity and death. According to the data released by the World Health Organization, there were approximately 14 million new cancer cases and 8.2 million cancer-related deaths in 2012. According to the 2012 data released by the National Cancer Registry Center, there are about 3.5 million new cases in China every year, accounting for about one-fifth of the global incidence; the number of cancer deaths is about 2.5 million, accounting for about one-quarter of the global cancer deaths. Although my country has made great progress in tumor surgery, chemical drug treatment, radiotherapy and biotherapy technology, the population base makes my...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC12N15/1068C12Q2525/191
Inventor 赵明玉陈威
Owner 北京圣谷同创科技发展有限公司
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