Development And Validation Of A 6-Dye STR Kit For The Analysis Of Degraded Forensic Samples

Research Background:Since the 1990s,STR-based DNA testing technology has been widely used in the field of forensic forensics due to its technical advantages of high sensitivity,high discriminatory capacity and ease of database construction,and has now become one of the most important technical tools in modern forensic science.However,with the development of DNA testing technology and its application in practice,a series of problems associated with the degraded DNA biological samples have become increasingly apparent.Forensic practitioners often have to deal with a variety of difficult degraded samples in the field,and the DNA in these biological samples are susceptible to degradation and break into small fragments due to exposure to a variety of physical,chemical and biological factors,resulting in a significant reduction in detection rates.Degraded DNA detection has been a hot and difficult research problem in the field of DNA in forensic science.The current mainstream forensic DNA analysis technology is the use of commercial kits for core STR detection,however,the longest amplification fragments of existing imported and domestic DNA testing reagents are mainly concentrated in the range of 400-500bp,and the ability to test such degraded samples has not been able to achieve a fundamental breakthrough.Compared with traditional STR technology,mini-STR technology is more suitable for DNA detection of degraded samples.mini-STR genotyping technology can design primers closer to the flanks of the STR repeat region,which can effectively reduce the fragment length of the amplification product,thus further improving the amplification sensitivity and genogenotyping success rate.However,studies have shown that the binding region of the mini-STR primer is closer to the repeat region and has a higher rate of base mutation,which may lead to site loss and other phenomena.At the same time,the presence of base deletions or insertions between the binding region of the mini-STR primer and the binding region of the traditional STR primer may lead to inconsistent genotyping results.In view of this,there is an urgent need to carry out research on a new STR fluorescence amplification system for DNA testing of degraded specimens,i.e.,on the basis of in-depth research on existing conventional STR kits and mini-STR kits,and in view of the latest technological development in related fields at home and abroad and the latest demand for forensic DNA testing reagents from front-line field testing units,to carry out research on 6-Dye DNA testing reagents.Based on the latest technological development in the related fields at home and abroad and the latest demand for forensic DNA testing reagents from frontline testing units,we will carry out research on key technologies of 6-Dye DNA testing reagents and develop fast,efficient and stable DNA testing reagents for case samples.Research Objective:1.Development and validation of a 6-Dye STR kit with strong stability for the analysis of degraded forensic samples.2.It is suitable for the detection of trace samples and degraded samples,and has strong anti-interference ability of inhibitors.Research Method:1.In this research,using multiplex ampli-fication technology and six-color fluorescence detection technology,16 CODIS loci and sex-determining loci（Amelogenin）were selected and labeled with six fluorescent dyes:FAM,HEX,TAMRA,ROX,NH618 and NH650.By redesigning primers and constructing an optimal composite amplification system,the developed STR kit is named STRscan-17LC.At the same time,Allelic Ladder,Spectral Calibration Reagent,Panel and Bin files corresponding to the kit were constructed.2.In this research,according to the specific requirements of ISFG and SWGDAM,the species specificity,precision and accuracy,sensitivity,stability,anti-inhibitor and other properties of the STRscan-17LC kit were verified.Population genetics studies were performed on 176 Beijing Han Chinese and 198 African Sierra Leone populations.Results:1.In this research,a six-color fluorescence composite amplification kit containing 16 autosomal STR loci and Amelogenin was developed,and the length of the amplified product fragment was less than330bp.2.An artificial degraded sample model was constructed for the STRscan-17LC reagent.The STRscan-17LC kit is more suitable for the detection of degraded DNA samples through the comparative study with the commercial kit Identifiler^?Plus.According to the specific requi-rements of ISFG and SWGDAM,the species specificity,precision and accuracy,sensitivity,stability,anti-inhibitor,consistency and other proper-ties of the STRscan-17LC kit were verified.The experimental results showed the STRscan-17LC kit has good stability,good species specificity,high sensitivity and strong anti-inhibitor interference ability.3.The better Allelic Ladder,Spectral Calibration Reagent,Panel and Bin files corresponding to the STRscan-17LC kit were constructed,and the appropriate Size Standard was selected.4.The STRscan-17LC kit was used to conduct population genetics research on 176 Beijing Han Chinese and 198 African Sierra Leone population.The experimental results showed that the STRscan-17LC kit is suitable for Beijing Han Chinese and African Sierra Leone population.5.Using the STRscan-17LC kit to directly amplify hair samples with follicles,oral swabs and FTA blood cards,etc.,complete genogenotyping was obtained.Conclusion:1.A six-color composite detection system of 17 STRs including 16CODIS STRs and 1 sex-determining STR was constructed,and the amplified product was within 330bp.Corresponding Allelic Ladder,Spectral Calibration Reagent,Panel and Bin files were constructed and appropriate Size standard was selected,which could well perform allele identification,separate spectral,electrophoresis data genogenotyping and molecular weight labeling.2.The constructed STR kit has strong stability,high sensitivity and good species specificity,can directly amplify case samples,and can be well used for the identification of degraded samples.3.Test results on Beijing Han Chinese and African Sierra Leone samples indicate that the kit can be used in both groups.