Development of simple and accurate detection systems for Cannabis sativa using DNA chromatography.

In recent years, the need for analyzing cannabis DNA has increased in order to accommodate the various types of cannabis samples encountered in forensic investigation. This study was aimed to establish a simple and accurate cannabis DNA detection system using DNA chromatography. Two chromatography chip systems with different features were successfully developed. One system (the "four-line version") involves tetraplex PCR amplification, which could be used to detect cannabis DNA and distinguish between drug-type and fiber-type cannabis using the tetrahydrocannabinolic acid synthase gene sequence. The other system was the "three-line version" with triplex amplification, which was specialized to distinguish cannabis from other plants, and had a sensitivity (10fg DNA/reaction) that was 100 times greater than the four-line version. In both versions, no false positives were observed for 60 medicinal plants, and accurate detection could be performed for several simulated forensic samples such as cannabis leaves, buds, stems, roots, seeds, resin, and cannabis leaves blended 1/100 in tobacco. Detection could be performed by the naked eye and only a thermal cycler was required for operation. Thus, DNA chromatography systems for cannabis detection are expected to contribute to the analysis of cannabis DNA in forensic chemistry laboratories without extensive equipment.

Determination of ABO genotypes by real-time PCR using allele-specific primers.

ABO grouping of biological specimens is informative for identifying victims and narrowing down suspects. In Japan and elsewhere, ABO grouping as well as DNA profiling plays an essential role in crime investigations. In the present study, we developed a new method for ABO genotyping using allele-specific primers and real-time PCR. The method allows for the detection of three single nucleotide polymorphisms (SNPs) at nucleotide positions 261, 796, and 803 in the ABO gene and the determination of six major ABO genotypes. This method required less than 2 h for accurate ABO genotyping using 2.0 ng of DNA. This method could be applicable for rapid and simple screening of forensic samples.

Simultaneous determination of seven informative Y chromosome SNPs to differentiate East Asian, European, and African populations.

Identification of the population origin of an individual is very useful for crime investigators who need to narrow down a suspect based on specimens left at a crime scene. Single nucleotide polymorphisms of the Y chromosome (Y-SNPs) are a class of markers of interest to forensic investigators because many of the markers indicate regional specificity, thus providing useful information about the geographic origin of a subject. We selected seven informative Y-SNPs (M168, M130, JST021355, M96, P126, P196, and P234) to differentiate the three major population groups (East Asian, European, and African) and used them to develop forensic application. SNP genotyping was carried out by multiplex PCR reaction and multiplex single base extension (MSBE) reaction followed by capillary electrophoresis of extension products. This method can be used to assign a haplogroup from both degraded male DNA samples and DNA samples containing a mixture of female and male DNA through PCR primers that generate small amplicons (less than about 150 bp) and are highly specific for targets on the Y chromosome. The allelic state of each marker was definitively determined from a total of 791 males from the three major population groups. As expected, samples from the three major population groups showed Y-haplogroups common in the region of provenance: Y haplogroups C, D, and O for East Asians; IJ and R1 for Europeans; and AB and E for Africans.

Allele frequencies and haplotypes for 28 Y-STRs in Ovambo population.

Y-chromosomal 28 short tandem repeat (STR) loci were investigated in unrelated healthy individuals of the Ovambo population from Namibia (n=54). Sixteen Y-chromosome short tandem repeat (Y-STR) polymorphic loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, GATAH4, DYS437, DYS438, and DYS448) were analyzed using AmpFISTR Yfiler Polymerase Chain Reaction (PCR) Amplification Kit. DYS441-445 and DYS446, DYS447, DYS449, DYS450, DYS459a/b, DYS463 and DYS464a/b/c/d were investigated using a multiplex PCR system. Fifty-one haplotypes were identified in 54 Ovambos. The STR diversity values for Y-STRs loci ranged from 0.036 (DYS392) to 0.900 (DYS 385).

Forensic species identification based on size variation of mitochondrial DNA hypervariable regions.

In this study, two new systems for species identification were developed based on size variation of mitochondrial DNA hypervariable regions among animals: one was a conventional method using non-fluorescent primer sets and agarose gel electrophoresis and the other was an automatic method using fluorescent primer sets and capillary electrophoresis. DNA samples from 18 mammal, four birds, and 19 fish species were amplified using three primer sets specific for mammals, birds, and fishes, respectively. The differences in the sizes of the polymerase chain reaction (PCR) products, ranging from about 350 to 900 bp, permitted us to identify species. These systems were successfully applied to various specimens from several criminal cases. In unknown samples, which were different in size from reference DNA markers, sequencing of the PCR products and subsequent BLAST analysis helped to identify species. Furthermore, the sequence data provided us with information on individuals. Because these species identification methods are very simple, easy, rapid, and exact, they are useful in the field of forensic science.

Allele frequencies for 15 STR loci in Ovambo population using AmpFlSTR Identifiler Kit.

Allele frequencies of 15 short tandem repeat (STR) loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA were determined in unrelated individuals in the Ovambo population from Namibia (n=195). Amplification was performed using AmpFlSTR Identifiler Kit. For each locus 6-20 alleles were observed. For all 15 loci, the combined matching probability is 3.3x10(16) and the power of exclusion is 99.99986%. AmpFlSTR Identifiler detection system is a valuable tool for individual identification in Ovambo population.

Allele frequencies for nine STR loci in Ovambo population using AmpFlSTR Profiler Kit.

Allele frequencies for the nine short tandem repeat (STR) loci D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S820 were investigated in 195 unrelated Ovambo (Bantus) population from Namibia. AmpFlSTR Profiler Kit was employed for amplification. For each locus, 6-19 alleles were observed. Comparison between Ovambo population data and that of other African populations was performed. AmpFlSTR Profiler detection system is a useful tool for individual identification in Ovambo population.