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A technology for constructing high-throughput sequencing library of trace DNA samples

A sequencing library and high-throughput technology, applied in the field of high-throughput sequencing library construction of trace DNA samples, can solve problems such as inability to obtain effective amplification, normal fragmentation of the genome, influence and other problems

Active Publication Date: 2021-10-08
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of transposase cutting and interrupting library construction is easily affected by the quality of the sample, resulting in cutting failure, the genome cannot be fragmented normally, and the subsequent amplification cannot be effectively amplified.
However, the normal process of library construction cannot be used for library construction below 20ng. When the initial amount is 50ng, the success rate of library construction is only 30%, and the quality of the library is poor.

Method used

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  • A technology for constructing high-throughput sequencing library of trace DNA samples
  • A technology for constructing high-throughput sequencing library of trace DNA samples
  • A technology for constructing high-throughput sequencing library of trace DNA samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] 1. Fragmented DNA end filling

[0096]

[0097] In the PCR instrument 20 ℃, 5min

[0098] 2. ddH 2 Make up to 200 μl with O, extract with phenol, settle with ethanol, and resuspend in 20 μl Tris-HCl

[0099] 3. Add dA to the 3' end of DNA

[0100]

[0101] In the PCR machine 70 ℃, 30min

[0102] 4. ddH 2 Make up to 200 μl with O, extract with phenol, settle with ethanol, and resuspend in 10 μl Tris-HCl

[0103] 5. Add adapters to DNA ends

[0104]

[0105] overnight at 16°C in a PCR machine

[0106] 6. ddH 2 Make up to 200 μl with O, extract with phenol, settle with ethanol, and resuspend in 25 μl Tris-HCl

[0107] 7. Electrophoresis at 120V, 30min, for gel recovery of 200-500bp bands (QIAquick Gel Extraction Kit)

[0108] 8. Specific experimental steps for antibody enrichment.

[0109] 1) Take 20ul A / G protein magnetic beads (Mill ipore, 16-663) and wash them twice with buffer, then resuspend the beads with 500ul buffer;

[0110] 2) Add 2-5ug antibo...

Embodiment 2

[0136] The method is the same as in Example 1, except that unmodified λDNA is added as an auxiliary nucleic acid fragment.

[0137] The results show,

[0138] 1. A sequencing library can be successfully constructed.

[0139] 2. The loss rate is slightly higher than that of Example 1 of the present invention.

[0140] 3. The quality value of all bases is about 28.

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Abstract

The invention provides a technology for constructing a high-throughput sequencing library of a trace DNA sample. Specifically, the method of the present invention includes the steps of: (a) providing DNA fragments and auxiliary nucleic acid fragments; (b) adding adapters to both ends of the DNA fragments and auxiliary nucleic acid fragments; (c) using specific antibodies to capture and enrich the ends with adapters (d) using specific enzymes to remove the auxiliary nucleic acid fragments; (e) using specific primers to amplify the captured and enriched DNA fragments and auxiliary nucleic acid fragments in the step (c). to obtain amplified products. The method of the invention is more suitable for the library construction of trace samples from a small amount of sources; the library has good quality and no pollution; the cost is low and the universal applicability is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a technology for constructing a high-throughput sequencing library of trace DNA samples. Background technique [0002] With the continuous innovation of life science and technology, people's research on life science is getting more and more in-depth, from life phenomenon to internal mechanism to genetic material, and the research on genetic material is gradually extending from one-dimensional to two-dimensional in space to the three-dimensional structure. Nucleic acid is the carrier of genetic information of life, and nucleic acid usually exists in many complex forms. To unravel the mystery of life science, genomics research and epigenetics research are imperative. The emergence of second-generation sequencing technology has greatly reduced the cost of sequencing, and also greatly improved the sequencing speed while maintaining high accuracy. While the second-generatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 赵小东康亚妮胡丛霞徐伟
Owner SHANGHAI JIAOTONG UNIV