Study On SNP Genotyping Of The FFPET And The Situation Of DNA Of FFPET Formalin Fixed Different Time

BackgroundFFPET(Formalin Fixed and Paraffin Embedded Tissues)are very important individual archives which are commonly used for disease diagnose and scientific research in clinic pathology,forensic pathology and other subjects,so it would be a reliable key evidence for cases of insurance fraud or inheritance of property or medical disputes,and other civil or criminal cases.At present,the hosipitals and research organizations fix human organizations using ordinary 10% formalin, so the DNA of FFPET is highly degraded due to physical and chemical influence factors when the tissues were fixed and embedded.It is very difficult to get the stable and credible genotype.FFPET is one of the most difficult material and is a focus as well as difficult problem in forensic serology.The COmbined DNA Index System (CODIS-based)13 core STR locis commercial kits(Plus and Cofiler)are widely used to genotype for blood,saliva,epithelial tissue,hairy root,osseous tissue and other biological samples,but its use for FFPET,its are very difficult to get correct genotype.Research indicates that the DNA extracted from FFPET could be influenced by formalin fixed time,thickness of the slice and the dewax method.Degraded DNA which come from different tissues have great different. Optimizing the digestion condition of proteinase,simplizing the procedures of the DNA extraction,purifying the extracted DNA may improve the purity quotient and avoid extra physical damage to DNA,but couldn't fundamentally change the current situation of the genotyping of the FFPET;Improving the PCR protocols such as increasing the number of PCR cycles and applying nesting PCR could increase the PCR production or PCR specificity,but could not increase the success rate of genotyping.Now,more researchs focus on minimizing the length of the amplicon to overcome the genotyping difficulties for the DNA severely degraded of the FFPET,and to improve the success rate of genotyping.Then,Many big firms are falling over themselves to develop series of miniSTR kits for highly degraded DNA samples.These kits have been proved more effective than the traditional STR kits in genotyping of FFPET,but there still are lots of problems,such as allele or loci drop-out, imbalance peak,Pull-up peak and and so on.Along with the progress in HGP(human genome project),more and more SNPs have been discovered,which is numerous,low mutation rate and more suitability for achieving high throughput and automation,then SNPs have been regraded as the third generation of genetic mark with great application perspective.Most of all,there is no need to get intact DNA,and DNA fragment with 50-100bp may achieve the SNP genotyping.This caters to the characteristic of FFPET whose DNA has been highly degraded,and casts light on the problems with the genotyping of FFPET.ObjectiveInquire into the influence to the DNA of paraffin embedded tissues formalin fixed different time.Based on the short amplicon ASPCR technology,we applied the SNP genotyping system(rs1454361and rs2046361)with high heterozygosity whose amplicon was limited less than 100bp to genotype the FFPET. By comparing its results of genotyping with that of the traditional PCR-STR technology(D2S441,whose amplicon is relatively short within CODIS),we explored its feasibility and superiority. Then investigating the polymorphism of rs1454361 and rs2046361 in China Chongqing Han.This study included two parts:PART ONG STUDY ON SNP GENOTYPING OF THE FFPET AND INFLUENCE ON DNA FROM DIFFERENT FIXED TIMEMaterials and Methods1.The FFPET samples including lung tissues,liver tissues,skin tissues,skeletal muscle tissues and brain tissues which were got from three frozen bodies in the forensic department of Chongqing Medical University ,then been fixed 5d,10d,15d and embedded with routine methods in the laboratory,and 2ml blood samples got from the heart of the bodies were prepared at the same time.2.Estimate the quantity and the quality of DNA from FFPET with AGE.3.Apply and compare the short amplicon ASPCR system with the PCR-STR system on the genotyping efficiency of FFPET.Result1.According to the result of AGE and compared with DNA marker,most DNA fragment of FFPET is shorter than 200bp,and those from the tissues fixed 5d present intense fluorescence bands nearby 100bp-200bp. The brain tissues fixed 10d present intense fluorescence bands nearby 50bp,the other present nearby 100bp . The liver tissues fixed 15d present still intense fluorescence bands nearby 100bp,the other present nearby 50 bp.2.The PCR-STR technology failed to genotype of all FFPET samples.But the genotyping results of FFPET by the short amplicon ASPCR presented positive results :The tissues fixed 5d presented 26 success results,the tissues fixed 10d presented 13 success results , The tissues fixed 15d presented 3 success results .PART TWO STHDY ON POLYMORPHISMS OF RSL454361 AND RS2046361 IN CHOGNQING HAN GROUP Materials and Methods119 blood samples of Chinese han individuals(without kinship)in chognqing were prepared.Establish the SNP genotyping system based on the short amplicon ASPCR technology,and apply this system to the 119 blood samples.Then,the frequency of alleles and genotypes of rsl454361 and rs2046361 were calculated,and the Hardy-Weinberg equilibrium test were performed.All forensic indexes were calculated by PowerStatesV12.ResultWithin the 119 China han individuals(without kinship)in Chongqing,the frequencies of the three genotypes(TT,TA and AA)indicates: rs1454361:0.2689,0.4705,0.2605,the frequencies of two alleles(TandA)are 0.5042 and 0.4958; rs 2046361 :0.2437,0.5798,0.1765,the frequencies of two alleles(TandA) are 0.5336 and 0.4664.The result of the Hardy-Weinberg equilibrium test is: rs1454361:χ2=0.4108(p>0.05);rs2046361:χ2=3.2370(p>0.05)。According to the PowerStatesV12, rs1454361/ rs2046361H is 0.471/0.58,DP is 0.638/0.573,Pm is 0.362/0.427,PE is 0.163/0.267,PIC is 0.37/0.37。Conclusion1.The DNA of FFPET that formalin fixed different time was all highly degraded.The DNA fragment which was extracted by organic solvent after the FFPET had been deparaffinized in xylene was shorter than 200bp,and centred about 100bp,and if the amplicon was longer than 150bp,it would be very difficult to genotype by PCR-STR.2.The short amplicon ASPCR is more efficient than PCR-STR in genotyping of highly degraded DNA such as FFPET.3.Our study report the genotype frequency and allele frequency distribution of rsl454361 and rs2046361 in chognqing han group,and provide the necessarily data in forensic medicine.Rsl454361 and rs2046361 can be valuable genetic marks for personal identification and paternity testing.And it can be valuable for forensic usage.