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Method for rapidly preparing Sanger sequencing template

A sequencing and template technology, applied in the field of bioengineering, achieves the effects of high sequencing success rate, overcoming high cost, and easy operation

Active Publication Date: 2019-07-05
NANJING GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Currently, conventional Sanger sequencing template preparation requires two steps of shaking culture and plasmid extraction (such as alkaline lysis or commercial mini-extraction kits), which takes about 14 hours in total.

Method used

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  • Method for rapidly preparing Sanger sequencing template
  • Method for rapidly preparing Sanger sequencing template
  • Method for rapidly preparing Sanger sequencing template

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] This example compares the sequencing effects of preparing Sanger sequencing templates within four hours using three different rolling circle amplification methods for preparing sequencing templates:

[0029] A. NEB phi29DNA Polymerase Reagent (Product No. M0269S, this reagent provides some supporting reagents and experimental operation scheme for RCA reaction) (hereinafter referred to as "NEB Reagent (phi29DNA Polymerase)")

[0030]B. Cited from literature (Frank B. Dean., John R. Nelson., Theresa L. Giesler., and Roger S. Lasken. 2001. Rapid Amplification of Plasmid and Phage DNA Using Phi29DNA Polymerase and Multiply-Primed Rolling Circle Amplification. Genome Research. 1095-1099) of the RCA protocol (indicated below as "methods in the literature")

[0031] C. The optimized RCA scheme provided by this application (hereinafter referred to as "this application")

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Abstract

The present application provides a method for rapidly preparing a Sanger sequencing template. The method comprises the following steps: a sample containing circular DNAs is subjected to a denaturationtreatment; a denatured product is subjected to rolling circle amplification; and the rolling circle amplification product treated by alkaline phosphatase is used to remove residual dNTPs.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for rapidly preparing a Sanger sequencing template. [0002] technical background [0003] The Sanger (ie Sanger) sequencing method, namely the dideoxy chain termination method, is a classic method for nucleic acid sequencing. The basic principle is to introduce dideoxynucleotide (ddNTP) into the reaction system in which DNA polymerase uses sequencing target DNA as a template to synthesize a complementary chain, causing the complementary chain in synthesis to terminate at the target DNA nucleotide corresponding to the ddNTP, forming A series of chain termination fragments, and then use polyacrylamide gel electrophoresis to distinguish each DNA chain termination fragment with a length difference of only one nucleotide, and judge the target DNA nucleoside at the corresponding position of the target DNA according to the type of ddNTP at the end of each fragment Types of acid. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2531/125C12Q2521/525C12Q2535/101C12Q2527/125
Inventor 樊隆周敬波蒋浩君刘家栋吴政宪
Owner NANJING GENSCRIPT BIOTECH CO LTD
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