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Method for extracting DNA from formalin fixed hair

A formalin and hair technology, applied in DNA preparation, recombinant DNA technology, sugar derivatives, etc., can solve the problems of too long soaking time in antiseptic solution, reduced activity of active sites, difficulty in extracting genetic material, etc. Time for hair digestion, increased surface area, method efficient effect

Inactive Publication Date: 2011-04-27
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in archaeological and forensic identification, it is often found that the hair follicles have been lost too late, or the hair root has been destroyed due to the long soaking time in the embalming solution
It is relatively difficult to extract genetic material from the hair shaft
At present, there have been reports of genome extraction from fresh hair shafts, and reports of genome extraction from tissue specimens that have been temporarily soaked and fixed, but there is no successful example of genome extraction from hair shafts that have been soaked and fixed for a long time. The stem is keratinized tissue, the main component is protein, and part of the hair shaft is difficult to digest; on the other hand, formalin not only binds to the amino group of the protein, which reduces or loses the activity of the active site, but also promotes DNA- DNA cross-linking and DNA-protein cross-linking lead to DNA fragmentation and difficulty in extraction

Method used

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  • Method for extracting DNA from formalin fixed hair
  • Method for extracting DNA from formalin fixed hair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Take 20cm of the hair shaft 2cm away from the hair follicle from the hair fixed in formalin for 10 years. Wash with NaOH for 5 minutes, then wash with sterile 2.0% HCl for 5 minutes, then rinse with sterile distilled water for at least 3 times, then wash with 75% and absolute ethanol in turn, and dry it for later use.

[0010] Grind the dry hair in a homogenizer filled with 300 μl of sodium citrate repair solution with a pH of 6.0 and a concentration of 0.01 mmol / L at 2°C, until the dry hair fragments cannot be seen with the naked eye, and transfer the grinding solution to In a new EP tube with a sterilized volume of 2ml, the homogenizer was washed twice with an equal volume of fresh repair solution and transferred to the EP tube, and then repaired in a boiling water bath for 8 minutes. After the restoration was completed, when it was naturally cooled down to room temperature, an equal volume of absolute ethanol was added and mixed evenly, then left at room temperature ...

Embodiment 2

[0016] Take hair that has been fixed in formalin for 30 years, and cut 20cm from the hair shaft 2cm away from the hair follicle. Wash with 2.0% NaOH for 5 minutes, then wash with sterile 2.0% HCl for 5 minutes, then rinse with sterile distilled water for at least 3 times, then wash with 75% and absolute ethanol once respectively, and dry it for later use.

[0017] Preparation and repair treatment of repair samples: Grind the dry hair in a homogenizer filled with 300 μl of sodium citrate repair solution with a pH of 6.0 and a concentration of 0.01 mmol / L, so that the dry hair fragments cannot be seen with the naked eye. , transfer the grinding solution to a new EP tube with a sterile volume of 2ml, wash the homogenizer twice with an equal volume of fresh repair solution and transfer them to the EP tube, then repair in a boiling water bath for 15 minutes. After the restoration was completed, when it was naturally cooled down to room temperature, an equal volume of absolute ethan...

Embodiment 3

[0021] From Mui hair in the Qing Dynasty, Mui 300 years ago, 20cm of the hair away from the hair follicle was taken from the hair. Wash with sterile 2.0% NaOH for 5 minutes, then wash with sterile 2.0% HCl for 5 minutes, rinse with sterile distilled water for at least 3 times, wash with 75% and absolute ethanol in turn, dry and set aside .

[0022] Repair sample preparation and repair treatment: Grind the hair dry in a homogenizer filled with 300 μl of sodium citrate repair solution with a pH of 6.0 and a concentration of 0.01 mmol / L at 5°C until the dry hair fragments are invisible to the naked eye , transfer the grinding solution to a new EP tube with a sterile volume of 2ml, wash the homogenizer twice with an equal volume of fresh repair solution and transfer them to the EP tube, then repair in a boiling water bath for 5min. After the restoration was completed, when it was naturally cooled down to room temperature, an equal volume of absolute ethanol was added and mixed ev...

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Abstract

The invention discloses a method of extracting DNA from formalin-fixed hair stems. After washing and pretreatment, the hair stems are put into a homogenizer full of 0.01mmol / L sodium citrate solution for grinding under 0 to 10 DEG C, then put in boiling water bath for 5 to 15min and be restored; ethanol is added for stewing, then eccentrically collected and deposited; hair digestive fluid and proteinase K solution are added into the deposition, and continuously digested for 1 to 2 hours in water bath at the temperature of 37-57 DEG C; DNA is extracted after digestion. The invention restores hair stem samples so as to totally or partially recover the activity of the protein active site. In addition, the preparation of the digestive fluid takes the characteristics of hair stem crack into full consideration so as to greatly improve the efficiency of hair stem crack and shorten the time of hair digestion. The method is used to extract not only the genome in the hair stem soaked in formalin but also the genome in the ancient hair stem.

Description

technical field [0001] The invention relates to a method for extracting DNA from a special hair shaft. Background technique [0002] In the field of biomedicine, formalin is often used as antiseptic fixation for animal tissue specimens, medical cadavers and pathological tissue specimens. It has been reported in the literature that formalin can damage the genetic information of the fixed specimen tissue, and the longer the soaking and fixing time, the greater the damage to the DNA. [0003] The hair is divided into two parts, the hair shaft and the hair root. The main component is keratin, including fibrin and cystine-rich non-helical protein. It is this keratin structure that makes it non-perishable, so the remains of ancient tombs During the cleaning process, most of the remains of the tomb owners only remained hair and bones. At the same time, hair is also one of the commonly used evidence in forensic identification. When using hair for genetic material extraction, the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H21/04C12N15/10
Inventor 黄菊芳曾乐平陈旦童建斌王慧罗学港
Owner CENT SOUTH UNIV
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