Preliminary Study Of Using SSH-PCR Technology For STR Typing Of Mixed Blood

Mixed spot is contained two or more individual body fluids, secretions which can be divided into two categories:one is involved different body fluids or secretion from the same individual, such as mixed bloodstains; the other is contained different body fluids from different individuals such as mixed spot composed of semen and vaginal fluids. The profiling of a sample with several DNA sources depends on the proportion of each sources in the sample, the combination of genotypes, and the quantity of samples DNA that are amplified. It means that the degree of difficulty of the profiling of a mixed sample is not the same. Mixed sample that the percentage of lower components less than 5% or 1:19 usually can not be detected. AmpFlSTR kit suggest that the lower components of mixed samples should be at least greater than 35pg to get reliable typing results. To improve successful typing rate of the lower components of mixing sample is one of the most difficult problems of forensic workers, at the same time it also have important research value in the field of medical genetics. SSH-PCR (Suppression Subtractive Hybridization SSH) is a technology of screening and separation of differentially expressed genes which based on suppression PCR and subtractive hybridization combined with standardization established by Diatchenko, etc in 1996. The basic process is:extract the mRNA of two different cells and reverse transcript into cDNA; convert the cDNA fragment into the flat end by using restriction enzyme.The cDNA with adaptors that we studied will be as a driver and the other cDNA process two rounds of sub-drive hybrid and two PCR amplified. Then differentially expressed cDNA will be amplifed. This process obeys the ideal second-order kinetics of hybridization that reannealing process generating homohybrid cDNA is faster for the more abundant single-stranded cDNA than the low abundant single-stranded cDNA, so that the concentrations of high and low abundance cDNAs become roughly equal. The molecules contain long inverted repeats on the ends and form stable panhandle-like structures after each denaturation-annealing PCR step.The resulting panhandle-like structure cannot serve as a template for exponential PCR, because intermolecular annealing of longer adaptor sequences is both highly favored and more stable than intermolecular annealing of the much shorter PCR primers.This is the suppression PCR effect. In the standard system, over 1,000-fold enrichment for rare sequences can be achieved in a single round of SSH procedure and some mRNA expression of low abundance is expected to be detected.Because of the ability of enrichment of DNA sequence in different expression of SSH in the this study we use PCR-STR amplification products for target sequence and connect adaptors directly then through suppression subtractive hybridization and PCR amplification, and try to increased the success rate of lower components genotyping of mixed DNA samples providing a new way for the DNA analysis of mixed samples.Methods1, blood samples from the China University of Medical Department.2, different types of blood samples mixed in accordance with 1:3,1:5,1:7,1:9,1:19,1:29,1:39.Results1, Polyacrylamide gel electrophoresis of different types of blood samples mixed in accordance with 1:3,1:5,1:7,1:9,1:19,1:29,1:39 showed that when the mixing ratio higher than 1:9, it can not be observed clearly bands in the gel. This indicates that the use of polyacrylamide gel electrophoresis is not as sensitive as capillary electrophoresis which the detection limit is1:19.2, We use different concentrations of T4 DNA ligase to connect adaptor. After the connection test it shows that when the enzyme concentration 35U/μ1(0.28Weiss equivalent units), the connection procession is more effective than other concentrations of ligase.3, According to polyacrylamide gel electrophoresis test results, in the second hybridization high components were significantly inhibited and not found bands in the expected location(183bp+22 bp+20 bp= 225bp) while low components are amplified and found significant bands in expected position (171bp+22 bp+20 bp= 213bp).4, In different hybridization conditions, products of hybridization are different. The expected position (213bp) has no obvious bands. There are bands of different intensities above 300bp the lanes.ConclusionThe high components were significantly inhibited and low components were effectively enriched by using SSH-PCR for detection of mixed samples at the proportion of 1:9.