The Development Of MiniSTR Multiplex Amplification Kit

Objective: The forensic DNA typing of nuclear STR loci has widely used in the cases involving forensic identification and kinship testing. While in forensic casework, it is possible and quite likely that the DNA samples will be highly degraded due to exposure to environmental elements or natural contaminants that can result in loss of information at higher molecular weight STR loci. This loss of signal may be the causing a partial DNA profile with allele or even complete locus drop out. It will make the difficulty of DNA typing. Redesign the primers and reducing the size of the PCR products (miniSTR) is an established and highly effective method for improving the molecular analysis of highly degraded DNA samples. This method was used to aid identification of World Trade Center victims in 2001 and named miniSTR technique. Applied Biosystems has developed a miniSTR kit capable of amplifying 8 core STR loci and Amelogenin with reduced PCR product sizes in combined DNA index system (CODIS). But several of the CODIS core STR loci have a large number of repeats or wide allele ranges or the limb sequence in too complex to redesign the primers that are not optimal for generating small amplicons. In order to solve this difficulty and improve the efficiency of the system, it is very important to find some miniSTR loci besides the CODIS loci. So, the purpose of this project is to choose some loci which are suitable for the character of Chinese Han population and establish a new fluorescent multiplex miniSTR system. A series of validation experiments, such as the sensitivity, accuracy and species specificity, were performed for the miniSTR multiplex systems according to the recommendation of the Technical Work Group DNA Analysis Methods (TWGDAM). At last, we established two miniSTR multiplex systems which were suitable for the Chinese Han population and it would impulse the development of the home-made forensic DNA test kit. Methods:â‘ Using the primers set from Coble's, a total of 120 samples from unrelated individuals in Hebei Han population was tested with the 26 miniSTR loci. According the obtained genetic data, we choose 8 loci which have highly polymorphism and short fragment length to investigate in the other areas. PCR products were analyzed with gel electrophoresis and visualized by silver staining. The genetic datas and alleles of each locus were obtained.â‘¡We construct the miniSTR multiplex systems using domestic Taq DNA Polymerase and detect the results by ABI 310/3130 Genetic Analyzer. Fragment size determination and genotyping were done with GeneMapper v3.2. Optimize the multiplex amplification conditions according to the outcomes of the different Mg²⁺ concentration, different anneal temperature and different cycle times.â‘¢Using molecular clone and sequencing techniques to construct the standard allelic ladders and naming the allelic ladders according to the principles of the international society of forensic haemogenetics(ISFG). Compile the autotyping template according to the statistic results.â‘£A series of validation experiments, such as the sensitivity, accuracy, species specificity, stale blood spot and degraded DNA samples in the cases were performed for the miniSTR multiplex systems and the commercial kits.Results:â‘ We have successfully constructed two fluorescent multiplex miniSTR systems of group 1 and group 2. The group 1 system includes D10S1248, D2S441, D1S1677 and D9S2157 four loci. The group 2 system includes D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin five loci. Allele frequencies and forensic parameters of the eight miniSTR loci were investigated by the two systems in 300 Chinese Han populations. The accumulated power of discrimination and power of exclusion for the eight loci were 0.99999999228 and 0.98547130732, respectively.â‘¡The two systems were easy to optimize and using domestic Taq can obtain satisfied amplification results. They are low-cost but efficient systems.â‘¢Using molecular clone technique constructed 64 clone of the 9 loci. After adjusting the concentration of each locus, we established the two systems'the standard allelic ladders. According to the experimental data and results of the sequencing set the automatic analysis naming parameters and established the automatic classification named template of the two fluorescent multiplex miniSTR amplification systems. They can type the samples accurately.â‘£Forensic applied research proved that the systems have a good species-specific. In addition to the emergence of rhesus monkeys in the Amelogenin peak, the remaining 8 loci in 8 common animals was no specific peaks appear. The results of the same individuals in different organizations are consistent. The sensitivity of the systems was 0.125ng template DNA. After testing the aged samples and the degraded DNA mode, it was demonstrated that these miniSTR systems had a high success for analysis of degraded samples. They can be used to analysis the samples which the conventional amplification kit failure to typing. The systems proved to be used in forensic identification and paternity test through the actual cases. The results are same to the identification conclusions.Conclusions: Our research successfully constructed two fluorescent multiplex miniSTR amplification systems suitable for the Chinese Han population. These systems can be successfully used in the ABI 310/3130 detection platform to get the reliable data. The research of forensic genetics implies that they are easily- regulated, low cost multiplex systems with stable outcome, high sensitivity and good species specificity. Based on these advantages, they could be recommended to the practice of forensic genetics. Especially they can be used in the analysis of degraded DNA samples. They have great value for provide low cost, good performance fluorescent miniSTR multiplex systems to the domestic forensic DNA analysis.