Method for evaluating DNA quality of FFPE sample

A sample medium and high-quality technology, applied in the field of molecular biology, can solve the problems of poor quality, small number of FFPE samples, single detection target area, etc., and achieve the effect of accurate DNA quality grading, clear result bands, and multiple detection positions

Inactive Publication Date: 2018-06-01
ANNOROAD GENE TECH BEIJING +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FFPE samples often have problems of small quantity and poor quality
The quality of DNA obtained from FFPE samples of different quality varies greatly. Using the same library construction strategy for such DNA samples, such as the same starting amount or the same library construction method, will directly affect the yield of the library, seriously affecting the quality of the library. Unable to perform sequencing studies
Existing DNA quality evaluation methods for FFPE samples usually have fewer grades, such as only two grades, and the detection target area is single, so it is impossible to reasonably grade the DNA quality of FFPE samples, and it is also impossible to make recommendations for subsequent sample extraction of DNA fragments. library process to give reasonable guidance
Therefore, the existing FFPE sample DNA quality evaluation methods still need to be improved.

Method used

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  • Method for evaluating DNA quality of FFPE sample
  • Method for evaluating DNA quality of FFPE sample
  • Method for evaluating DNA quality of FFPE sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Evaluation experiment of DNA fragment quality in FFPE samples

[0050] 1. Primer Preparation

[0051] According to the sequence of the GAPDH gene as a template, primers P58-1, P58-2, P60-1 and P60-2 were designed, wherein, primers P58-1 and P58-2 were designed to amplify a fragment with a length of 236bp, and primer P60 -1 and PP60-2 were used to amplify a fragment with a length of 505bp. According to the sequence of the β-actin gene as a template, primers P02-1, P02-2, P04-1 and P04-2 were designed, wherein, the obligatory P02-1 and P02-2 were designed to amplify a fragment with a length of 733bp, Primers P04-1 and P04-2 were designed to amplify a fragment with a length of 1069bp, and primers P02-1 and P04-2 were designed to amplify a fragment with a length of 1789bp. The nucleotide sequences of the above primers are shown in Table 1.

[0052] Table 1

[0053]

[0054] 2. DNA extraction from FFPE samples

[0055] FFPE samples with storage times of 0.5...

Embodiment 2

[0071] Sample 1, sample 2, and sample 3 of Example 1 were used for database construction.

[0072] (1) Preparation of DNA fragments to be sequenced

[0073] Take sample 1, sample 2, and sample 3 500ng each for DNA fragmentation. The fragmentation condition is 30 cycles, 30 seconds ON, 30 seconds OFF, fragmented to a fragment size of about 200bp, and the double-stranded DNA fragment after fragmentation Some of these are blunt-ended, others contain 3' or 5' overhangs.

[0074] (2) Perform end repair to obtain blunt-ended DNA fragments.

[0075] A. The reaction system is as follows:

[0076]

[0077]

[0078] B. Incubate in a PCR instrument for 30 minutes at a temperature of 20°C;

[0079] C. The reaction product was purified with magnetic beads, and eluted with 19.5 μL of elution buffer EB (Elution Buffer) to obtain blunt-ended DNA fragments.

[0080] (3) Add A to the blunt-ended DNA fragments at the 3' end

[0081] A. The reaction system is as follows:

[0082]

...

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Abstract

The invention relates to a method for evaluating DNA quality of a FFPE sample. Generally, primers shown by nucleotide sequences, such as SEQ ID No:1-SEQ ID No:8 are used for carrying out PCR amplification of DNA in the FFPE sample; quality grades of DNA are effectively evaluated according agarose gel electrophoresis results, and accurate grading of DNA quality of the FFPF sample is carried out.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for evaluating DNA quality in FFPE samples. Background technique [0002] Formalin-fixed and Paraffin-embedded (Formalin-fixed and Paraffin-embedded, FFPE) processing method can preserve tissue for a long time or prepare tissue specimens required for testing, so in clinical pathological examination, tumor gene detection and medical scientific research are often used in the process. Worldwide, approximately billions of tissue samples are stored in hospitals or tissue banks. The vast majority of these were formalin-fixed and paraffin-embedded samples. The huge number of archived FFPE samples provides a valuable resource for retrospective studies, elucidation of disease mechanisms, discovery of therapeutic targets, and indication of prognosis. [0003] However, formaldehyde, as an important component of formalin, can cause extensive cross-linking between nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q1/6886C12Q2531/113C12Q2565/125
Inventor 刘伟王秀莉董超玄兆伶李大为梁峻彬陈重建
Owner ANNOROAD GENE TECH BEIJING
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