Double chain nucleic acid fragment joint adding method, library constructing method and kit

A double-stranded nucleic acid and library construction technology, applied in the field of genetic engineering, can solve the problem that the library construction efficiency of target nucleic acid fragments needs to be improved, and achieve the effects of simplifying the construction process, reducing the amount of nucleic acid input, and improving the sequencing coverage.

Active Publication Date: 2018-05-22
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the library construction efficiency of the ta...

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  • Double chain nucleic acid fragment joint adding method, library constructing method and kit
  • Double chain nucleic acid fragment joint adding method, library constructing method and kit
  • Double chain nucleic acid fragment joint adding method, library constructing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0288] Example 1 Construction of a paired library comprising two bubbling adapters

[0289] Figure 19 Depicted how to construct a paired library containing two bubbling adapters. The details are as follows:

[0290] 3ug DNA was fragmented using Covaris to obtain 200-1800bp fragments. The fragmented DNA was then size-selected using magnetic beads to retain fragments of 300-1000 bp with an average size of 650 bp. 500 ng or 1.2 pmol of size-selected DNA was used in library preparation. End repair was performed using T4PNK and T4 DNA polymerase to generate 5' phosphorylated blunt-ended fragments, and dA tailing was added to the fragments. The first bubble adapter Ad203 was ligated to the DNA fragment by A-T ligation. The ligation product was amplified by PCR using uracil-containing primers and PfuCx polymerase, which allows the presence of uracil in the template. With USER enzyme, uracil-specific excision reagent enzyme, uracil DNA glycosylase (abbreviated UDG) and a mixtur...

Embodiment 2

[0291] Example 2 Construction of a paired library comprising two L-oligonucleotide adapters

[0292] Figure 22 Depicted is a schematic diagram of the construction of a paired library comprising two L-oligonucleotide adapters.

[0293] 3ug DNA was fragmented using Covaris to obtain 200-1800bp fragments. The fragmented DNA was then size-selected using magnetic beads to retain fragments of 300-1000 bp with an average size of 650 bp. 500 ng or 1.2 pmol of size-selected DNA was used in library preparation. The fragmented DNA was end-repaired using shrimp alkaline phosphatase and T4 DNA polymerase to obtain dephosphorylated blunt-ended fragments. The first L-oligonucleotide adapter Ad169 was ligated to the DNA fragment in two steps. For the first step, a second oligonucleotide is ligated by blunt ends in the presence of a short helper oligonucleotide with a 3'-end modification. A "heat inactivation" step is used to inactivate the ligase and remove the helper oligonucleotide, t...

Embodiment 3

[0294] Example 3 Construction of a paired library comprising bubbling and clamp junctions

[0295] Figure 23 Depicted is a schematic diagram of the construction of a paired library comprising a bubble adapter as the first adapter and a clamp adapter as the second adapter.

[0296] 3 μg of DNA was fragmented using Covaris to generate 200-1800 bp fragments. The fragmented DNA was then size-selected using magnetic beads to retain fragments of 300-1000 bp with an average size of 650 bp. 500 ng or 1.2 pmol of size-selected DNA was used in library preparation. End repair was performed using T4PNK and T4 DNA polymerase to generate 5' phosphorylated blunt-ended fragments, and dA tailing was added to the fragments. The first adapter, bubble adapter Ad201, was ligated to the DNA fragment by A-T ligation. The ligation product was amplified by PCR using uracil-containing primers and PfuCx polymerase, which allows the presence of uracil in the template. With USER enzyme, uracil-speci...

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Abstract

The invention discloses a double chain nucleic acid fragment joint adding method, a library constructing method and a kit. According to the double chain nucleic acid fragment joint adding method, a 3'terminal lateral joint is connected to the 3' terminal of a double chain target nucleic acid fragment; the double chain target nucleic acid fragment comprises a connection site, the connection site comprises the 3' terminal, which contains 3'-hydroxyl, the connection site is an incision, notch, or a 5' terminal embossment; the 3' terminal lateral joint comprises the 5' flat terminal and non-connection 3' terminal of 5'-phosphoric acid; and the 3' terminal lateral joint connection method comprises a step of adopting ligase to connect a double chain target nucleic acid fragment and the 3' terminal lateral joint. According to provided method, the 3' terminal lateral joint is connected to the 3' terminal of a double chain target nucleic acid fragment; based on the method, a library is established, the library is applied to cPAL and synthesis sequencing, and is suitable for genomic sequence or whole exon sequencing; the primary amount of nucleic acid for library construction is reduced, the process for library construction is simplified, the sequencing covering rate of a GC enriched area is improved, and the sequencing performance is enhanced.

Description

technical field [0001] The present application relates to the field of genetic engineering, in particular to a method for adding adapters to double-stranded nucleic acid fragments, a library construction method and a kit. Background technique [0002] Large-scale genome sequence analysis is key to understanding a variety of biological phenomena. Therefore, based on the demand for low-cost, high-throughput sequencing or individual genome resequencing, the development of new target nucleic acid fragment library construction methods has been promoted, and at the same time, the research on new sequencing methods for parallel analysis of multiple target nucleic acid fragments has been promoted. However, the library construction efficiency of the target nucleic acid fragment library still needs to be improved. Contents of the invention [0003] The purpose of this application is to provide a new method for adding adapters to double-stranded nucleic acid fragments, a library con...

Claims

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Application Information

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IPC IPC(8): C12P19/34C40B50/06
CPCC12P19/34C40B50/06
Inventor 江媛拉多杰·德马纳克埃文·贺罗维茨安德烈·阿莱克谢耶夫赵霞阮婕
Owner MGI TECH CO LTD
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