Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method and kit for rapid construction of single-cell DNA sequencing library

A DNA sequencing, single-cell technology, applied in the field of single-cell DNA sequencing library construction, can solve the problems of complex process, unsuitable for large-scale production applications, poor genome coverage and uniformity, etc.

Active Publication Date: 2015-10-07
HANGZHOU BERRYGENOMICS GENE DIAGNOSIS TECH
View PDF8 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned single-cell DNA sequencing library construction methods and kits mainly have two defects: First, the process from WGA to library construction is complicated and takes a long time. It takes at least two days to complete the entire process, which is not suitable for Large-scale production applications; secondly, the PCR base bias introduced by WGA amplification is relatively large, resulting in poor coverage and uniformity of the entire genome

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
  • Method and kit for rapid construction of single-cell DNA sequencing library
  • Method and kit for rapid construction of single-cell DNA sequencing library
  • Method and kit for rapid construction of single-cell DNA sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The construction method of the single-cell DNA sequencing library of Example 1 mainly includes the following steps (see image 3 ):

[0079] (1) Cell lysis: This step can be performed using any protease, protein denaturing reagent and lysis buffer system known to those skilled in the art that are suitable for cell lysis.

[0080] (2) Pre-amplification of genomic DNA can be performed using any thermostable DNA polymerase known to those skilled in the art, for example: LA-Taq, rTaq, Phusion, Deep Vent, Deep Vent (exo-), Gold 360 , Platinum Taq, KAPA 2G Robust.

[0081] The preamplification primers used in the present invention are mainly composed of three parts: a common region, which has different sequences in different sequencing platforms; a degenerate base region, which only contains two kinds of bases, A and C, or A and G , or T and C, or T and G; a region of random degenerate bases, where each base can be A, T, C, or G, and two sulfurs are added between the last t...

Embodiment 2

[0114] The library construction method in Example 2 is basically the same as in Example 1, the difference is that in Example 2, the starting sample is multi-cell rather than single-cell (please refer to image 3 ).

[0115] A specific example of performing multicellular DNA sample sequencing according to the method of Embodiment 2 of the present invention is shown below

[0116] Step 1: Multiple cell lysis. Prepare cell lysis buffer as shown in Table 1, incubate at 60°C for 20 minutes, 95°C for 4 minutes, then keep samples at 4°C.

[0117] Table 1

[0118]

[0119] Step 2: Genomic DNA pre-amplification. Prepare the pre-amplification PCR system shown in Table 2.

[0120] Table 2

[0121]

[0122] The PCR reaction scheme is: a.95°C for 3 minutes→b.16 cycles as follows: 98°C for 20 seconds, 15°C for 50 seconds, 25°C for 40 seconds, 35°C for 30 seconds, 65°C for 40 seconds, 72°C for 1 minute→ c. Keep at 4°C.

[0123] Step 3: Secondary amplification of genomic DNA. Pr...

Embodiment 3

[0132] The library construction method in Example 3 is basically the same as in Example 1, except that in Example 3, the starting sample is DNA instead of cells. In this example, the starting sample can be genomic DNA, mitochondrial DNA, long PCR products, or long chromatin-precipitated DNA (see image 3 ).

[0133] A specific example of performing DNA sample sequencing according to the method of Embodiment 3 of the present invention is shown below

[0134] Step 1: Fragmentation of long fragments of DNA. Prepare cell lysis buffer as shown in Table 1, incubate at 60°C for 20 minutes, 95°C for 4 minutes, then keep samples at 4°C. Alternatively, DNA fragments were directly incubated at 95°C for 4 minutes, and then the samples were kept at 4°C.

[0135] Table 1

[0136]

[0137] Step 2: DNA pre-amplification. Prepare the pre-amplification PCR system shown in Table 2.

[0138] Table 2

[0139]

[0140] The PCR reaction scheme is: a.95°C for 3 minutes→b.16 cycles as follo...

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 method and kit for construction of a single-cell DNA sequencing library, specifically to a method and kit for construction of a single-cell DNA sequencing library used for high-flux sequencing.

Description

technical field [0001] The present invention relates to a method and a kit for constructing a single-cell DNA sequencing library, and more specifically, the present invention relates to a method and a kit for constructing a single-cell DNA sequencing library for high-throughput sequencing. Background technique [0002] With the completion of the Human Genome Project, Sanger sequencing has entered the research of many researchers. However, the continuous innovation of technology has made this dideoxynucleotide-terminated sequencing mode increasingly unable to meet the needs of sequencing, so a new high-throughput sequencing (also called second-generation sequencing) mode emerged as the times require. This sequencing technology enables lower sequencing costs, higher sequencing throughput, and shorter sequencing time. The core idea of ​​high-throughput sequencing technology is sequencing-by-synthesis, that is, to determine the sequence of DNA by capturing the markers of newly ...

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): C40B50/06C12N15/11
Inventor 石燕滨吴凯王文璐张建光
Owner HANGZHOU BERRYGENOMICS GENE DIAGNOSIS TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com