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Sanger sequencing reaction optimization agent, sequencing reaction system and sequencing method employing optimization agent

A sequencing reaction system and sequencing reaction technology, applied in the field of molecular biology, can solve the problems of short effective sequencing sequences, interrupted sequencing signals, and inaccurate sequences.

Inactive Publication Date: 2015-04-08
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to secondary structures or other intrinsic factors, complex structures are a great challenge for DNA sequencing
Common special DNA structures include hairpin structure, high GC content sequence, repetitive sequence, etc. In the conventional sanger sequencing method, such structures often cause the sequencing signal to be interrupted in advance, and the signal weakens quickly, resulting in short effective sequence and inaccurate sequence. question

Method used

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  • Sanger sequencing reaction optimization agent, sequencing reaction system and sequencing method employing optimization agent
  • Sanger sequencing reaction optimization agent, sequencing reaction system and sequencing method employing optimization agent
  • Sanger sequencing reaction optimization agent, sequencing reaction system and sequencing method employing optimization agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Sequencing case of hairpin DNA sequence (ShDNA sequence, hairpin structure) using the sequencing method of the present invention

[0030] 1. In this embodiment, the PCR reaction system for sequencing the hairpin DNA samples consists of the following:

[0031] 5×sequencing buffer: 2μl

[0032] Template (150ng / μl): 1μl

[0033] Primer (3.2μM): 2μl

[0034] Sanger sequencing reaction optimizer 1 μl (by volume: 1 part of Bigdye, 0.2 part of dGTP-BigDye, 0.2 part of 10 μM dGTP and 0.2 part of 10 μM dCTP)

[0035] Water: 4 μl.

[0036] 2. The PCR reaction procedure for sequencing the hairpin DNA samples in this embodiment is as follows:

[0037] Pre-denaturation at 98°C for 8 minutes; quenching in ice water;

[0038] Take 98°C for 10 seconds, 50°C for 1 minute, and 60°C for 4 minutes as a cycle, and perform 40 cycles;

[0039] Store at 4°C.

[0040] 3. The steps of carrying out ethanol / sodium acetate purification to the PCR reaction product:

[0041] (1) Add...

Embodiment 3

[0070] Example 3 Sequencing case of sequences with high GC content using the sequencing method of the present invention

[0071] 1. In this embodiment, the PCR reaction system for sequencing samples with high GC content is composed of:

[0072] 5×sequencing buffer: 2μl

[0073] Template (150ng / μl): 1μl

[0074] Primer (3.2μM): 2μl

[0075] Sanger sequencing reaction optimizer 1 μl (by volume: 1 part of Bigdye, 0.5 part of dGTP-BigDye, 0.5 part of 10 μM dGTP and 0.5 part of 10 μM dCTP)

[0076] Water: 4 μl.

[0077] 2. The PCR reaction procedure for sequencing the samples with high GC content in this embodiment is:

[0078] Pre-denaturation at 98°C for 8 minutes; quenching in ice water;

[0079] Take 98°C for 10 seconds, 50°C for 1 minute, and 60°C for 4 minutes as a cycle, and perform 40 cycles;

[0080] Store at 4°C.

[0081] 3. Steps for purifying the PCR reaction product:

[0082] (1) Add 1 μl 125mM EDTA to the bottom of each tube, and add 1 μl 3M NaAc to the bottom...

Embodiment 4

[0089] Example 4. Comparative test example: Sequencing comparison of sequences with high GC content using conventional sequencing methods

[0090] This example is a comparative test example of Example 3, that is, sequences with high GC content are sequenced by conventional methods.

[0091] 1. PCR reaction system using conventional methods:

[0092] 5×sequencing buffer: 2μl

[0093] Template (150ng / μl): 1μl

[0094] Primer (3.2μM): 2μl

[0095] BDT: 1 μl

[0096] Water: 4 μl

[0097] 2. PCR program using conventional methods:

[0098] 95°C for 5 minutes; quenching;

[0099] 40 cycles were performed with 95° C. for 10 seconds, 50° C. for 1 minute, and 60° C. for 4 minutes as one cycle.

[0100] Store at 4°C.

[0101] 3. Steps for purifying the PCR reaction product:

[0102] (1) Add 1 μl 125mM EDTA to the bottom of each tube, and add 1 μl 3M NaAc to the bottom of each tube;

[0103] (2) Add 25 μl of 100% alcohol to each tube, mix well, and let stand at room temperature...

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Abstract

The invention relates to the technical field of molecular biology and discloses a Sanger sequencing reaction optimization agent, as well as a sequencing reaction system and a sequencing method employing theoptimization agent. According to the volume ratio, the Sanger sequencing reaction optimization agent provided by the invention comprises the following components: 1 part of BigDye, 0.2-1 part of dGTP-BigDye, 0.1-1 part of dGTP and 0.1-1 part of dCTP. The optimization agent is added into the Sanger sequencing reaction and is applied to sequencing of common special DNA structures such as a hairpin structure, a high-GC sequence or a repetitive sequence, and the problems that the sequencing signal is interrupted in advance in the sequencing process, the effective sequencing sequence is short due to fast signal weakening and the sequence is inaccurate in the sequencing process can be effectively solved, so that a reliable and accurate sequencing result relative to the complex structure can be provided.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a Sanger sequencing reaction optimization agent, a sequencing reaction system and a sequencing method using the optimization agent. Background technique [0002] In molecular biology research, DNA sequence analysis is the basis for further research and modification of target genes. The Sanger dideoxy chain termination method (Chain Termination Method) invented by Frederick Sanger is a commonly used DNA sequencing method. The Sanger sequencing method is based on the nucleotide starting at a fixed point, randomly introducing ddNTPs labeled with four different colors of fluorescence to terminate the extension reaction, thus forming a large number of extension products with fluorescently labeled ends and different lengths , and then use high-resolution capillary gel electrophoresis to separate these extension products. By distinguishing the four different fluorescent color...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869
Inventor 孙子奎丁方美王锋高文学方钰
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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