Free DNA library construction method and detection method for low and medium frequency mutation in free DNA

A technology of DNA library and construction method, which is applied in the direction of chemical library, microbial measurement/testing, biochemical equipment and methods, etc., which can solve the problems of complex detection methods and high cost, and achieve the goal of overcoming sequencing errors and amplification errors, and retaining mutations The effect of frequency

Active Publication Date: 2016-08-03
元码基因科技(苏州)有限公司
View PDF9 Cites 13 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires secondary amplificatio

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Free DNA library construction method and detection method for low and medium frequency mutation in free DNA
  • Free DNA library construction method and detection method for low and medium frequency mutation in free DNA
  • Free DNA library construction method and detection method for low and medium frequency mutation in free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Detection method of cfDNA low-frequency mutation in plasma

[0071] 1. Sample extraction

[0072] Use the free DNA extraction reagent to extract the free DNA in the subject's plasma. The main steps are as follows: temporarily store the whole blood sample in ice at 4°C, turn on the high-speed refrigerated centrifuge, put the blood collection tube at 4°C and centrifuge at 1600g for 10 minutes to remove residual cells, and then centrifuge at 16000g at 4°C After 10 minutes, remove residual cells and transfer the supernatant to a new 2 mL centrifuge tube.

[0073] Add proteinase K600μL fully dissolved and mixed plasma in a 2mL centrifuge tube, vortex shaker to mix for 10s, and briefly centrifuge.

[0074] Add 600 μL of the mixed buffer GBmix, and centrifuge briefly; place in a water bath at 56°C for 10 minutes; take out the centrifuge tube, cool at room temperature for 5 minutes, and centrifuge briefly; add 300 μL of frozen absolute ethanol, gently invert to mix,...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a free DNA library construction method. A 3' end of a single-stranded free DNA is connected with a sole sequence tag; the sole sequence tag comprises a random sequence and a linker sequence; an upstream primer and a downstream primer for PCR amplification are added for PCR amplification; the upstream primer is a primer designed aiming at a target low frequency mutation; the downstream primer is the primer corresponding to the linker sequence. In addition, the invention further relates to a detection method for low and medium frequency mutation in a free DNA. The 3' end of the single-stranded free DNA is connected with the sole sequence tag; the upstream primer and the downstream primer for PCR amplification are added for PCR amplification; one end of each of the upstream primer and the downstream primer is connected with the sequence tag respectively; the amplified DNA is sequenced online on an Illumina platform. According to the method, detection on cfDNA low frequency mutation can be simply and efficiently achieved; the target of fetal inheritance or tumor cell mutation detection can be achieved through cfDNA detection.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a free DNA library and a method for detecting low-frequency mutations in the free DNA. Background technique [0002] Cell-free DNA (CellFreeDNA, referred to as cfDNA) refers to free DNA in peripheral blood, which exists in the form of naked nucleic acid, mainly derived from fragmented DNA produced during apoptosis or cell lysis (such as necrosis caused by physical stimulation). or immune cell killing), the size of the DNA fragments produced after the reaction is basically around 160-180bp. [0003] As early as 1947, Mandel et al. found free DNA in the serum and plasma of healthy people, but it did not attract enough attention at that time. In 1977, Leon et al. found that there was a certain relationship between tumor and free DNA content in serum. Afterwards, researchers detected oncogene mutations in the plasma and serum of tumor patients, which were c...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C40B50/06C12Q1/68
CPCC12Q1/6869C12Q1/6883C12Q1/6886C12Q2600/156C40B50/06C12Q2535/122C12Q2525/191C12Q2525/179
Inventor 田埂
Owner 元码基因科技(苏州)有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap