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Reagent and kit for rapidly extracting nucleic acid from FFPE sample and application of reagent and kit

A technology for extracting nucleic acids and samples, applied in the field of molecular biology, can solve the problems of unsatisfactory binding effect of binding buffer, low nucleic acid yield and purity, and achieve the effect of convenient nucleic acid extraction operation, improved binding effect and simple formula

Active Publication Date: 2021-11-02
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the binding effect of the binding buffer used in the above method is still not ideal, and the yield and purity of the nucleic acid obtained by coordinating with the lysis, washing and elution buffer are low

Method used

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  • Reagent and kit for rapidly extracting nucleic acid from FFPE sample and application of reagent and kit
  • Reagent and kit for rapidly extracting nucleic acid from FFPE sample and application of reagent and kit
  • Reagent and kit for rapidly extracting nucleic acid from FFPE sample and application of reagent and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] The reagents for rapidly extracting nucleic acids from FFPE samples in this embodiment include dewaxing agents, lysis buffers, proteinase K, binding buffers, magnetic beads, nucleic acid precipitation aids, washing buffers and elution buffers;

[0093] The dewaxing agent is n-hexadecane;

[0094] The lysis buffer comprises the following components: SDS 40mM, Tween 20 0.6% (v / v), Tris-H 2 SO 4 40mM, the pH value is 8.0, and the balance is water;

[0095] The proteinase K is a commercially available product with a concentration of 1.5 mg / mL;

[0096] The binding buffer comprises the following components: guanidine isothiocyanate 1M, Tween 20 0.6% (v / v), Triton X-100 4% (v / v), EDTA 40mM, KCl 0.2M, Tris-HCl 40mM, the pH value is 8.5, and the balance is water;

[0097] The magnetic beads are commercially available silanol magnetic beads, and the concentration used is 0.5 mg / mL;

[0098] The nucleic acid precipitation aid is ethanol, and the use concentration is 25% (v / ...

Embodiment 2

[0126] The reagents for rapidly extracting nucleic acids from FFPE samples in this embodiment include dewaxing agents, lysis buffers, proteinase K, binding buffers, magnetic beads, nucleic acid precipitation aids, washing buffers and elution buffers;

[0127] The dewaxing agent is n-hexadecane;

[0128] The lysis buffer comprises the following components: SDS 15mM, Tween 20 0.2% (v / v), Tris-H 2 SO 4 15mM, the pH value is 8.5, and the balance is water;

[0129] The proteinase K is a commercially available product with a concentration of 1.5 mg / mL;

[0130] The binding buffer comprises the following components: guanidine isothiocyanate 4M, Tween 20 0.2% (v / v), Triton X-100 2% (v / v), EDTA 10mM, KCl 0.1M, Tris-HCl 20mM, the pH value is 8.5, and the balance is water;

[0131] The magnetic beads are commercially available silanol magnetic beads, and the concentration used is 0.5 mg / mL;

[0132] The nucleic acid precipitation aid is ethanol, and the use concentration is 20% (v / ...

experiment example

[0147] The DNA content and OD260 / 280 and OD260 / 230 values ​​of the DNA extracted in Examples 1-8 and Comparative Examples 1-4 were detected by a spectrophotometer NanoDrop One, and the results are shown in Table 2.

[0148] Table 2 DNA content and OD260 / 280, OD260 / 230 detection values:

[0149]

[0150]

[0151] Adopt Agilent 2100 bioanalyzer to carry out qualitative analysis to the DNA that embodiment 1 extracts, the result is as follows figure 1 shown. From figure 1 It can be seen that the distribution of DNA fragments extracted in Example 1 is mainly between 2400-34000bp.

[0152] As can be seen from the above experimental results, the binding buffer of the present invention is combined with Tween series surfactants (such as Tween 20 or Tween 40) and polyethylene glycol octylphenyl ether (Triton X-100), Cooperating with other components, it significantly improves the binding effect of nucleic acid and magnetic beads. On the one hand, it increases the binding amount...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a reagent and a kit for rapidly extracting nucleic acid from an FFPE sample and application of the reagent and the kit. The combination buffer solution in the reagent is simple in formula and low in cost, the combination effect of the nucleic acid and the magnetic beads can be remarkably improved, and the combination amount of the nucleic acid is increased. According to the method, the lysis buffer solution, the binding buffer solution and the washing buffer solution with specific components are combined for use, tissue cells are fully lysed, protein bound with nucleic acid is digested, the nucleic acid is completely released, then the released nucleic acid is specifically bound to the magnetic beads, the binding amount of the nucleic acid is increased, effective separation of the nucleic acid and other lysates is achieved, the magnetic beads combined with the nucleic acid are washed for multiple times, impurities such as protein and lipid are thoroughly removed, and the nucleic acid isfinally eluted from the magnetic beads to obtain high-purity and high-yield nucleic acid. The reagent / kit is simple in composition, low in cost and convenient for nucleic acid extraction operation, and the extraction time can be greatly shortened.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a reagent, kit and application for rapidly extracting nucleic acid from FFPE samples. Background technique [0002] With the impact of precision medicine on pathology and the continuous upgrading of research methods, pathologists, in addition to paying attention to traditional tissue morphology, study FFPE (Formalin Fixation and Paraffin Embedding) samples at the molecular level And more and more. Most of the FFPE samples are related to tumors, so the study of these tissue samples provides a favorable basis for exploring new tumor classification, diagnosis and prognosis standards, developing experimental techniques for early detection of tumors, translational medicine and individualized medicine. [0003] Although FFPE samples allow long-term preservation of tissues, their special production methods and storage methods will also affect nucleic acid molecule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 崔国鹏董超张银张晓亮杨增利
Owner AUTOBIO DIAGNOSTICS CO LTD
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