| IntroductionABO blood group is the first genetic system of human blood that ever be found. In 1990, the nucleic acid sequence of ABO gene and the base sequence of the alleles that decided the ABO blood group were first reported by Yamamo-to, et al. It is the base of ABO genotype detection at DNA level. ABO genoty-ping techniques used at present include PCR - restriction fragment length polymorphism (PCR - RFLP) , single - stand conformation polymorphism (SSCP) , PCR — direct sequencing ,allele - specific amplify ( ASA) ,consumed allele -specific primer analysis ( CASPA) ,etc. Among them, the ASA is more sensitive,simple and rapid, therefore that has been widely applied in forensic identification. The CASPA method choosed in this study is a new method of ABO gen-otyping based on ASA method. This method has at least three advantages. Firstly, the CASPA method requires amplification in only one tube and the subsequent steps are performed by an automated procedure using a capillary sequencer. Therefore, the method is more simple, rapid, and sensitive. Secondly, this method does not require a long running time for electrophoresis since the allele - specific primers are very small as compared products. Thirdly, it can be applied to the typing of SNPs of a very low molecular weight DNA template that has been decomposed because a PCR fragment needs not necessarily be extended to an ordinary chain length in this method. This study is based on the CASPA method, but the primer had been modified, and we also studied the applicability of this method in forensic identification.MaterialsSix allele - specific primers at different length labeled by fluorescent dyes 6- FAM and HEX, one no - labeled community primer. DNA samples which ABO gene type had already been known. Blood stains of 146 Han unrelated individuals. Mixed stains from rapes cases, cigarette butts, sweat stains, muscles, cartilages, thighbones and hair roots.MethodsDNA was isolated from samples by the phenol - chloroform method, some samples were extracted by the chelex - 100 synchronously, we designed six allele - specific primers at different length based on the nucleotide positions 261, 297 and 803 of the exon 6 and 7 of ABO gene (two specific primers per nucleotide position ) , four of the primers were labeled with fluorescent dyes 6 - FAM, others with fluorescent dyes HEX. The community primer was not labeled. The best condition of PCR reaction was tested by the method of grouping experiment and compound experiment. ABO genotype of DNA template adopted had been known. The separate condition of the amplified products by sequencer is; PCR products was diluted 100 times. 1μl of dilution was mixed with 50μl of 95% formamide and 1μl of Size Stander LIZ -120, denatured at 95 ℃ for 5min and cooled in ice water for 10min. The mixture was analyzed by a model 3100 sequencer with 47cm length capillary column (POP4) for 20min at 15KV. The CASPA typing patterns of ABO gene were produced by Gene Scan Software. The veracity of the analysis results was validated by stability test and elution test. The ABO genotyping results of 146 samples were analyzed by X2 test. We also detected the sensitivity, practicability of the method in forensic area, and detected the extracted DNA templates by two different methods.ResultsOur results showed that the best condition and system of PCR reaction is: PCR was performed using 20 ul mixture containing 50mmol KCl, 10mmol Tris -HCl (PH 8. 3), 1. 5mmol MgCl2, 0. 01mg/mlgelation,200μmol each dNTP, 1U of Ampli Taq Gold polymerase,0. 25ug BSA, 2pmol of primer R297 G, 4pmol of other primers (L261A,L261G, R297 A,R803C,R803G) ,and 20ng of genomic DNA. The condition of PCR amplification is:95℃ for 11min, and then 50 cycles of 95℃ for 20s, 55℃ for 20s and 72℃ for 10s. After the last cycle the samples were incubated at 4℃. The results of this study accorded with that of stability test and elution test. Nine genotypes AAb,AB,AO1,BOv,O1Ov,AA, BB,O1O1,BO1 were discovered in 146 Han unrelated individuals. The results of genotype frequencies showed that P >0. 25 and the Dp value was 0.766. The results of sensitivity test showed that the least DNA detected was 10ng. The results of contrasting test of two DNA extractive method showed that the ABO genoty-ping method we established can be used to detect DNA extracted by phenol -chloroform method.DiscussionIn this study, six allele - specific primers at different length were designed. They were labeled with two different fluorescent dyes on basis of the nucleotide positions 261, 297 and 803 of the exon6 and 7 sequences of ABO gene. Six kinds of allele ( A,Ab,B,O1,Ov,O2)and twenty -one ABO genotypes can be distinguished by this method. This method has some advantages as follow; Six allele - specific primers were used in one PCR reaction to amplify three SNPs of the ABO gene, the genotype was determined by the detection of the remainder primers. Therefore, the method is simple and inexpensive. Furthermore , the time of extension was shortened because the remainder primers is what to be detected and the amplified fragments is short. Therefore, the period of test is short. No additional control sample was required because the LIZ -120 was mixed with samples as size stander when PCR products were separated by electrophoresis , and the patterns were analyzed by Gene Scan Software. The results of 146 samples detected by this method accorded with that of PCR - ASA method and elution test. So the stability and repetition of this method is good. The sensitivity of this method is also high for the lowest dose of DNA that can be detected is only 10ng. In one word, the method is simple, rapid, nicety and sensitivity in the test of ABO genotype and can be widely used in the detection of crimi-...
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