High-sensitivity pyrosequencing reaction liquid and preparation method thereof

A preparation method and sequencing reaction technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of difficult access to enzymes, and achieve the effects of increasing optical signal intensity, low cost, and improving sensitivity

Inactive Publication Date: 2012-12-26
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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AI Technical Summary

Problems solved by technology

The pyrosequencing system using PPDK can achieve the detection of fmol-level templates by increasing the amount of luciferase, but there is no commercial PPDK supply on the market, and the enzyme is not easy to obtain through genetic engineering expression

Method used

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  • High-sensitivity pyrosequencing reaction liquid and preparation method thereof
  • High-sensitivity pyrosequencing reaction liquid and preparation method thereof
  • High-sensitivity pyrosequencing reaction liquid and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of highly sensitive pyrosequencing reaction solution

[0039] 1. Optimization of ATP sulfurylase concentration

[0040] It is known that the Michaelis constant Km of the reaction of APS and ATP sulfurylase to form an enzyme-substrate complex is 0.56 μmol / L. In order to achieve the maximum reaction rate, and the excess substrate will not inhibit the enzyme activity, the substrate concentration needs to be 10 times of the Km value, so the fixed APS concentration in this experiment was 5 μmol / L. Other components in the pyrosequencing reaction solution are: 0.1mol / L Tris-HAc (pH 7.7), 2mmol / L EDTA, 10mmol / L Mg(Ac) 2 , 0.1% BSA, 1mmol / LDTT, 0.4g / L PVP, 0.4mmol / L D-luciferin, 5μmol / L APS, 18U / ml Klenow, 1.6U / ml apyrase, 29.2mg / L luciferase. Prepare 6 reaction solutions in parallel, and add 0.1, 0.2, 0.5, 1, 2, and 5 μL of ATP sulfurylase (Sigma, 20 U / ml, 13.3 μmol / L) to each 100 μL sequencing reaction solution, that is, ATP sulfuration The enzyme con...

Embodiment 2

[0045] Example 2: Typing of avian influenza virus by pyrosequencing

[0046] 1. cDNA sample

[0047] One H5N1 avian influenza virus cDNA sample was provided by the Jiangsu Provincial Center for Disease Control and Prevention; three H9N2 avian influenza virus cDNA samples (A / HeNan / 2 / 04, A / HeNan / 3 / 04 and A / HeNan / 5 / 04) Provided by the Medical Department of Beijing Agricultural University.

[0048] 2. Gene-specific PCR amplification

[0049] The M gene and HA gene of avian influenza virus H5 and H9 subtypes were amplified by gene-specific PCR, respectively. The 100 μL PCR reaction system contains 0.2 μmol / L of each primer (see Table 1), 0.5 mmol / L of dNTP, MgCl 2 3.0mmol / L, 1× buffer, 2.5U Taq DNA polymerase and 100-500ng cDNA template. The thermal cycle parameters are: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 10 seconds, annealing at 60°C (M gene) or 55°C (HA gene) for 20 seconds, extension at 72°C for 40 seconds, and 30 cycles of amplification;...

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Abstract

The invention relates to a preparation method of high-sensitivity pyrosequencing reaction liquid. In the invention, a background signal can be controlled in a lower level under the condition of high-density luciferase by increasing the ATP sulphonated enzyme quantity to restrict the reaction between a subtract APS and the luciferase, and a reaction signal remarkably raises, therefore, the basic-group sequencing can be carried out by using dozens of fmol quantity of DNA which is one magnitude order less than that of the usual one. Therefore, for a sample with seldom sources and a product with low amplification efficiency, the accurate short-sequence measurement can be realized, and meanwhile, the novel high-sensitivity reaction liquid can be also applied to a portable bioluminescent analyzer which takes a photosensitive tube array with low price as a detector, thereby realizing fast, high-efficiency and low-cost DNA analysis.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and relates to a high-sensitivity pyrosequencing reaction solution and a preparation method thereof. Specifically, by increasing the amount of adenosine triphosphate sulfurylase and luciferase in the pyrosequencing reaction solution, the pyrosequencing reaction signal is significantly increased At the same time, its background signal is controlled at a very low level. Background technique [0002] DNA sequence determination technology is one of the core technologies of modern life science research. The currently widely used DNA sequence analysis techniques are basically based on the dideoxynucleotide chain termination method (Sanger method), but the Sanger method can only be qualitative but not quantitative; it is suitable for large-scale sequencing and is not suitable for determining single-base differences and cannot be accurately determined The base sequence behind the primer. Pyrosequencing t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/66
Inventor 周国华武海萍吴文娟
Owner HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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