The Further Research Of DNA Extraction Method For Bone And Teeth

Objectives:The purpose of this project is to conduct DNA detection of bones and teeth in forensic case, through in-depth study of different decalcifying agent, digestive juice, analysis and comparison of the skeletal DNA concentration using SPSS software, looking for the most suitable decalcifying agents and digestive juices, to establish a set of extraction method,the extraction method is simple, accurate and reliable results, and high efficiency.Methods:1.We use different acid decalcification for DNA extraction of skeleton(10%hydrochloric acid decalcification, 10% nitric acid decalcification, Nitric acid and formic acid decalcification, Formic acid decalcification, the modified HAS decalcifying fluid),decalcified, digestion, purification, amplification, the 3130 xl to analyze DNA genetic analyzer;2.We use the EDTA decalcify fluid of different concentrations(0.25mol/L, 0.5mol/L,1mol/L) for decalcifying bone meal, each group of decalcification time according to the number of small to large, 3h, 6h, 12 h, 24 h,respectively. Experimental process after decalcification, digestion, purification, determining the concentration of the DNA and performing STR analysis of the extracted DNA and finally to find the most suitable concentration and action time of the EDTA decalcified.3.We use three different digestive juices(CTAB, ipads incubation buffer, Investigator QIAamp DNA) on Bone digestion, purification, determination of DNA concentration and SPSS statistical analysis software, and amplification, and 3130 xl genetic analyzer forDNA analysis, find the most suitable digestive juices.4. We use three different digestive juices(CTAB, ipads incubation buffer, Investigator QIAamp DNA) for teeth digestion, purification, amplification, the 3130 xl to analyze DNA genetic analyzer, Our purpose is to verify the feasibility of the experiment.Results:1.Acid has a strongdecalcification, but also has large damage to the DNA,so there was very little amount of DNA.2. In 3-24 h period, bone DNA by three different concentration of EDTA decalcified liquid level was present a different trend after decalcification, 0.25 mol/L group showed a trend of increase, in 12 to 24 h level was the highest, 16 STR loci all detection rate is62.5%; 0.5 mol/L set a trend of increase first, then decrease, in 6 and 12 h level was the highest, 16 STR loci all detection rate is 90%; 1 mol/L group showed a trend of decrease,in 3 to 6 h level was the highest, 16 STR loci all the detection rate of 67.5%. When decalcified time consistent and EDTA decalcified liquid concentration is not at the same time, 0.5 mol/L group got the DNA density in different time periods are higher than other two groups;3.One gram of bone get the amount of DNA was 26.53±5.47 ng/μL, 23.63±4.56ng/μL, 14.93±3.88 ng/μL,respectively, by Bone incubation buffer, DNA Investigator Kit and CTAB method. Single factor analysis of variance showed statistically significant between the three groups of data. The results show that the loci detection rate and RFU value is roughly the same by bone incubation buffer method and DNA Investigator Kit method after PCR amplification and electrophoresis detection. The two methods were better than CTAB method.4. Using CTAB, Bone incubation buffer, DNA Investigator three kit extraction methods for DNA test was performed on the teeth, the results show that the less the amount of DNA obtained by the CTAB method, and the other two methods had obvious difference.Conclusions:1.hydrochloric acid, nitric acid, formic acid, salicylic acid and acetic acid is widely used in clinical medicine. Both these acid could be used as calcium supplements.After bone demineralization PCR multiplex amplification,not typing results obtained, We can thus considered acids are not suitable for extraction of forensic field of skeleton, teeth DNA;2.In the 3-24 hour period, bone meal showed different trends by three EDTA decalcify fluid, 0.25mol/L EDTA decalcifying fluid group showed increasing trend, 0.5mol/L EDTA decalcifying fluid group showed increased at first and then decreased, 1mol/L EDTA decalcifying fluid groups show a decreasing trend, 0.5 mol/L EDTA decalcified liquid DNA level was highest 6 to 12 hours, show that moderate concentration of decalcified fluid is crucial, the concentration is too low or too high are adverse to the DNA extraction, decalcified time control in between 6-12 hours is appropriate.3.Analysis of skeletal DNA concentration was obtained using CTAB, Bone incubation buffer, DNA Investigator kit of the three kinds of extraction methods by SPSS statistical software, the results show that there exists a significant difference between the three methods of DNA, CTAB method to get the amount of DNA is less, it clear difference with the other two methods, Differences between the two groups in statistical sense,but DNA amplification effect is not very different.4.In the process of the practical cases,the bone incubation buffer method and DNA investigator kit method are both considered as the optimization of absence For DNA extraction of bone and teeth.