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DNA sequencing reaction reagent and preparation method thereof

A DNA sequencing and reaction reagent technology, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of low enzyme efficiency, inaccurate sequence, and short effective sequencing sequence, and achieve the effect of accurate sequencing results

Pending Publication Date: 2019-08-06
通用生物(安徽)股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The efficiency of enzymes currently used in sequencing reactions is not high, and the efficiency of polymerases needs to be improved, which leads to problems such as early interruption of sequencing signals and rapid signal weakening during sequencing, resulting in short effective sequences for sequencing and inaccurate sequences.

Method used

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  • DNA sequencing reaction reagent and preparation method thereof
  • DNA sequencing reaction reagent and preparation method thereof

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Embodiment 1

[0047] A DNA sequencing reaction reagent prepared from BigDye v3.1, 5X Buffer and coenzymes;

[0048] Wherein the volume ratio of BigDye v3.1, coenzyme and 5X Burr is 1:1:7, after mixing BigDye v3.1, coenzyme and 5X Buffer evenly according to the ratio, the DNA sequencing reaction reagent is obtained;

[0049] The BigDye v3.1 is a product produced by Thermo Company;

[0050] The coenzyme is selected from 0.5-1U Taq (F667Y) DNA polymerase;

[0051] The concentration of the coenzyme is 96.3%;

[0052] The enzyme activity of the coenzyme is 5.1U / ul;

[0053] The preparation of described coenzyme comprises the steps:

[0054] S1. Gene synthesize the corresponding target sequence and load it into the corresponding target vector, and transform the obtained plasmid into the corresponding expression strain;

[0055] S2. Picking a single clone for expression screening;

[0056] S3, amplifying and expressing the screened bacterial strain by IPTG induction;

[0057] S4, carry out p...

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Abstract

The invention discloses a DNA sequencing reaction reagent and a preparation method thereof. The reagent is prepared from BigDye v3.1, 5X Buffer and a coenzyme, wherein the volume ratio of the BigDye v3.1 to the coenzyme to the 5X Buffer is (1-1.2) to (1-1.1) to (7-8.2). According to the DNA sequencing reaction reagent disclosed by the invention, the coenzyme is added to a reagent added during a sequencing reaction, and the compounding ratio of the added reagent is adjusted; in a Sanger sequencing reaction, through the actual situation of the DNA sequencing reaction, the DNA sequencing reactionreagent can be applied to sequencing of familiar special DNA structures such as a hair pin structure, high GC content sequence or a repeated sequence, the problems that a sequencing effective sequence is short and sequencing is inaccurate caused by the conditions that sequencing signals are discontinued in advance and signals are quick to diminish in the sequencing process can be effectively solved, and a reliable and pinpoint sequencing result over against complex structure DNA can be provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA sequencing reaction reagent and a preparation method thereof. Background technique [0002] There are currently two main methods for DNA sequencing: the chemical method of Maxam and Gilbert and the dideoxynucleotide termination method of Sanger, in which the Sanger method uses a chain terminator-similar to 2′,3′-dideoxynucleosides of normal dNTP Triphosphate specifically terminates the extended DNA chain, and its reaction is often applied to a modified T7 bacteriophage DNA polymerase, ie, sequenase, in the sequencing process. [0003] Many characteristics of natural polymerases are not suitable for DNA sequencing. The characteristics that sequencing enzymes should possess include: (1) High processivity: processivity refers to the extent to which the chain continues to extend before the enzyme detaches from the primer-template annealing complex ; (2) heat resistance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2521/101C12Q2531/113C12Q2535/101
Inventor 雍金贵黄小锋施荣辉
Owner 通用生物(安徽)股份有限公司
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