Denaturation method of sequencing reaction product for Sanger method

A sequencing reaction and product technology, applied in the field of denaturation of sequencing reaction products, can solve problems affecting sequencing quality, multi-time cost, labor cost, and consumption

Inactive Publication Date: 2021-03-26
武汉康圣达医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During denaturation, the hydrogen bond that maintains the stability of the double helix is ​​broken, and the stacking force between bases is destroyed, but it does not involve changes in its primary structure. Sequencing on the machine needs to keep the fragments single-stranded, otherwise the sequencing quality will be seriously affected
[0004] The current common method is to denature the sequencing reaction product at 95°C for 3 minutes, and then cool it down; PCR instrument or water bath and ice cubes are required, which may generate some harmful gases, and require a lot of time and labor costs.

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  • Denaturation method of sequencing reaction product for Sanger method
  • Denaturation method of sequencing reaction product for Sanger method
  • Denaturation method of sequencing reaction product for Sanger method

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preparation example Construction

[0032] The preparation and purification methods of the sequencing reaction products are summarized as follows:

[0033] 1. Use the pre-designed primers for amplifying the target fragments, and use human genomic DNA as a template to carry out PCR reaction under certain conditions to amplify a large number of target fragments.

[0034] 2. Use enzymatic hydrolysis to remove the primers and dNTPs in step 1 to facilitate the preparation of subsequent sequencing products.

[0035] 3. Use single-stranded sequencing primers, buffer, BigDye and the enzymatic hydrolysis product in step 2 to obtain single-stranded DNA with ddNTPs of different sizes.

[0036] 4. Use 125mM EDTA solution, 3M sodium acetate solution (pH 5.2), and 75% ethanol to remove impurities in step 3 to obtain single-stranded DNA with ddNTPs of higher purity and different sizes.

[0037] 5. Hydrogen chains may be formed between single-stranded DNA with ddNTPs of different sizes, which can be separated by high-temperatu...

Embodiment 1

[0038] Embodiment 1 is used for the denaturation method of the sequencing reaction product of Sanger method

[0039] In the prior art, the sequencing reaction products based on the Sanger method are denatured at high temperature first, and then cooled in an ice bath, which requires the use of a large number of PCR instruments or water baths, which wastes a lot of ice cubes and may generate some harmful gases, which costs more Shortcomings such as time and manpower, according to the characteristics of the reagent HiDi, this embodiment uses the existing instruments in the laboratory to be directly sequenced on the machine after shaking, which reduces the generation of harmful gases caused by operating links and high temperatures, and saves operating time and unnecessary Waste of necessary manpower and material resources. The specific method is as follows:

[0040] Add 10 μL of HiDi to the sequencing reaction product in the 96-well plate (about 30 ng of DNA in each well after na...

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Abstract

The invention provides a denaturation method of a sequencing reaction product for a Sanger method. The denaturation method comprises the following steps of adding deionized water or deionized formamide into the sequencing reaction product for the Sanger sequencing technology in a 96-well plate, putting the 96-well plate on an oscillator, fully oscillating, and directly sequencing on a machine after oscillating. According to the characteristics of a reagent HiDi, an existing instrument in a laboratory is changed into a mode of directly sequencing on the machine after oscillation, so that the generation of harmful gas caused by operation links and high temperature is reduced, the operation time is saved, and unnecessary waste of manpower and material resources is reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a denaturation method for sequencing reaction products of the Sanger method. Background technique [0002] Sequencing technology is the most important detection method in molecular biology, the gold standard for gene polymorphism and gene mutation detection, and the reference standard for many other technical methods. The advantage of sequencing technology is that it can directly measure the gene sequence of the sample, obtain the accurate information of gene variation, and can detect multi-site mutations or unknown mutations of a certain DNA sequence, which is widely used in the detection of individualized treatment of tumor-targeted drugs , genetic disease gene mutation detection, blood disease gene mutation detection, HLA typing and matching, pathogenic microorganism drug resistance detection and many other fields. [0003] The first generation of DNA sequencing techniques was th...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/101C12Q2527/125C12Q2565/125
Inventor刘丽琼徐海霞陈然郝玮李小青
Owner武汉康圣达医学检验所有限公司