Study On SNP Genotyping Of The FFPET By Short Amplicon ASPCR

IntroductionFFPET(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. However, the CODIS-based 13 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 when it comes to FFPET, these kits are very difficult to be stable and credible to get the genotype, because the DNA of FFPET is highly degraded due to physical and chemical influence factors during the tissues were fixed and embedded.Research indicates that the DNA extracted from FFPET could be influenced by formalin fixed time, thickness of the slice and the dewax method. 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 don't contribute to increase the success rate of genotyping. Now, more research locus 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, artefactual peak, imbalance peak, sutter band 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 regarded 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.Based on the short amplicon ASPCR technology, we established a SNP genotyping system (rs1454361) with high heterozygosity whose amplicon was limited less than 100bp. After investigating the polymorphism of rs1454361 in China Liaoning Han, we applied the system to genotype the FFPET, By comparing its results of genotyping with that of the traditional PCR-STR technology (D8S1179,whose amplicon is relatively short within CODIS), we explored its feasibility and superiority, then developed a new way for genotyping of the highly degraded DNA.Materials and Methods1. The FFPET samples including lung tissues, skeletal muscle tissues and brain tissues which were got from three frozen bodies, then been fixed 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; In addition, 152 blood samples of Chinese han individuals (without kinship) in Liaoning province were prepared.2. Establish the SNP (rs1454361) genotyping system based on the short amplicon ASPCR technology, and apply this system to the 152 blood samples. Then, the frequency of alleles and genotypes of rsl454361 were calculated, and the Hardy-Weinberg equilibrium test were performed. All forensic indexes were calculated by PowerStatesV12. 3. Estimate the quantity and the quality of DNA from FFPET with ultraviolet spectrophotometer and AGE.4. Apply and compare the short amplicon ASPCR system with the PCR-STR system on the genotyping efficiency of FFPET.Result1. Within the 152 China han individuals (without kinship) in Liaoning province, the frequencies of the three genotypes (TT, TA and AA) are 0.2632, 0.4671 and 0.2697, the frequencies of two alleles (TandA) are 0.4967 and 0.5033. The result of the Hardy-Weinberg equilibrium test is: x²=0.6571 (P>0.05) . According to the PowerStatesV12, H is 0.467, DP is 0.640, Pm is 0.360, PE is 0.160 and PIC is 0.375.2. All FFPET samples were detected by ultraviolet spectrophotometer, and their OD₂₆₀/OD₂₈₀ about 1.6-1.8. According to OD₂₆₀ the DNA concentration of 4-piece group is about 140μg/ml-1g/ml, that of 1-piece group is about 20-80μg/ml.3. According to the result of AGE and compared with DNA marker, most DNA fragment of FFPET is shorter than 200bp, and those from the lung tissues and the skeletal muscle tissues present intense fluorescence bands nearby 100bp. Otherwise, those from the brain tissues present intense fluorescence bands nearby 50bp. With the same volume, the fluorescence intensity of the 4-piece group is obviously much stronger than that of 1-piece group.4. The PCR-STR technology failed to genotype of all FFPET samples. But the genotyping results of FFPET by the short amplicon ASPCR presented 7 positive results in 4-piece group expect two from brain tissues, and 4 positive results in 1-piece group.Conclusion1. The DNA of FFPET that formalin fixed 7 days was 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 in Liaoning han group, and provide the necessarily data in forensic medicine. Rsl454361 can be valuable genetic marks for personal identification and paternity testing. And it can be valuable for forensic usage.