Discussion on the extraction method of trace DNA evidence
Trace amounts of DNA often exist in potential forms in the physical evidence of the case, such as clothing, bricks, etc. Although the success rate of DNA testing for micro DNA evidence is low, once it is successfully obtained, it can be solved. Direct evidence, therefore, for potential DNA evidence, the extraction of samples is the key to the success of DNA testing. The extraction of DNA evidence is different from the extraction of other physical evidence. In order to facilitate the extraction and purification of the next step, it is generally necessary to transfer the sample. The extraction of sample DNA is roughly divided into two types: 1 direct clipping 2 other extraction tools Direct clipping For the extraction of trace DNA evidence, direct clipping should be the best extraction method to avoid DNA loss during the transfer process, but the sample should be carefully analyzed to highlight the key parts, and the multi-band light source and blood mark detector can be used. Find the location of DNA attachment and spot extraction to increase the DNA extraction success rate. Other tools For non-cuttable samples, we need to optimize the extraction method to maximize DNA extraction efficiency and avoid detection failure caused by DNA loss. 1 Wiping method At present, the commonly used wiping extraction method is a two-step method, that is, after repeatedly wiping with a wet cotton swab, and then wiping with a dry cotton swab until the surface carrier is dry, and then using two cotton swabs for DNA extraction. Some customers use Copan flocking swabs instead of conventional cotton swabs. Since the flocking swabs are made of nylon fiber filling technology, the nylon fibers are placed on the carrier perpendicular to the substrate, which can maximize the efficiency of DNA collection and elution. The special basket has a laboratory verification to increase the efficiency of nucleic acid extraction by 60% , which is especially suitable for the extraction of trace DNA evidence. 2 Vacuum adsorption method The vacuum adsorption method is divided into dry and wet. The dry adsorption method directly absorbs the DNA evidence on the surface of the sample and collects it for DNA detection. The principle of the wet adsorption method is to firstly clean the sterile liquid under a certain pressure. Spray on the surface of the sample, and then use the effect of vacuum adsorption to collect the liquid carrying the DNA evidence, and separate the liquid and DNA evidence to obtain the potential DNA evidence in the sample. Since wet adsorption utilizes the penetration and dissolution of liquids, the extraction efficiency of DNA is better due to the dry adsorption method, especially for porous and textile samples. Ultra-micro DNA material extractor   Applicable to conventional methods for extracting failed samples   Particularly suitable for porous materials such as clothing, bricks, etc.   Can realize large-area sample extraction   The extraction efficiency can be several times to several tens of times of the conventional method. There are many influencing factors in the micro DNA test, and a slight inadvertent result will result in unsatisfactory test results. Therefore, to improve the success rate of micro-DNA testing, it is necessary not only to work hard on the scientific extraction of the materials, but also to timely check and select appropriate testing strategies and programs.
Preparation of Fenebute
1. Prepare the first intermediate
Benzaldehyde and ethyl acetoacetate into ethanol, stir at room temperature under the catalysis of organic alkali reaction with filter cake filtration after 45 ~ 50 h, after the completion of the filter with ethanol elution filter cake, dry cake solids, then ethanol filtrate concentrated to a quarter of the original volume, to cold, concentrate filtering alcohol washing, solid, merging two solid is the first intermediate; The mole ratio of benzaldehyde, ethyl acetoacetate and ethanol is 1:2.5 -3:12-15.
2. Prepare the second intermediate
The first intermediate is added to the mass concentration of 20% sodium hydroxide solution, and the reaction is stirred at 85-90°C for 2-5 hours. After filtration, the filter cake is used for filtration. After filtration, the filter cake is washed, the filtrate is combined and cooled, and the filtrate is cooled by stirring for 3h. Solid second intermediate; The mass ratio of the first intermediate to the sodium hydroxide solution is I: 1.8-2.0;
3. Prepare the third intermediate
The second intermediate was dissolved in pure acetic anhydride solution for reflux 3 ~ 5h. After the reflux, the acetic anhydride was concentrated and dissolved with toluene. After the dissolution, ammonia water was added, and the reaction took place at 60_65°C for 1 ~ 1.5 h. Filtrate, wash and dry the precipitated solid to get the solid third intermediate; The mass ratio of the second intermediate, pure acetic anhydride solution, toluene and ammonia water is 5:1-1.2:0.5-0.8:0.5-0.8;
4. Prepare Finebute
The third intermediate was dissolved in 25% sodium hydroxide solution and cooled to _5°C ~ 10°C. After cooling, sodium hypochlorite was added slowly within 15-20min for 1h reaction, followed by ice bath reaction of 0.5 ~ 1.5 h and water bath reaction of 0.5 ~ 1.5 h. Finally, the temperature was gradually increased to 60 ~ 80°C for 1-5 h, and the reaction was cooled again after completion. After cooling, hydrochloric acid was added in the ice water bath to adjust the PH to 2, and the decolorization was stirred at room temperature. After filtration, sodium hydroxide was added in the ice water bath to adjust the PH to 6-7, and the cooling was stirred for 2 hours. The mass ratio of the third intermediate, sodium hydroxide solution and sodium hypochlorite is 1.0:0.8-1.0:1.0-1.1.
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