We compared automated DNA extraction instruments of AutoMate Express, EZ1 Advanced XL, Maxwell 16 and QIAcube for forensic purpose. DNA was extracted from fresh bloodstains and fresh diluted bloodstains on cotton and denim, three 3 mm punches of FTA card containing buccal cells collected by EasiCollect, and head hair roots and shafts. The genomic and mitochondrial DNA was quantied by real-time PCR assay using D17Z1 locus and/or the C region of hyper variable region 1 (HV1). The extracted DNA was used to amplify 15 STR loci of Identiˆler kit and/or the A and the C regions in HV1.AutoMate Express tended to give the highest DNA concentration from all the samples except for FTA cards and hair roots. Full STR proˆles were obtained using all the instruments from bloodstains on cotton and denim, FTA cards and hair roots. Out of the 15 diluted bloodstain samples on cotton and denim, full STR proles were obtained from 14 and 15 samples for AutoMate Express, 11 and 14 samples for EZ1 Advanced XL, 0 and 6 samples for Maxwell 16, and 8 and 14 samples for QIAcube, respectively. When 20 ml of the denim extract was concentrated and ampliˆed with 1 ng of 9947A DNA, PCR inhibition was slightly observed using AutoMate Express and Maxwell 16, but not observed when using EZ1 Advanced XL and QIAcube.When the A and the C regions of HV1 were ampliˆed using 2,000 copies of the hair root DNA extracted by all the instruments, positive bands were observed from almost all the samples. On the other hand, when 2,000 copies or less of the hair shaft DNA were ampliˆed, the bands of the A region were weaker than those of the C region for all the instruments especially for AutoMate Express.In conclusion, AutoMate Express and EZ1 Advanced XL are suitable for DNA

Ultraviolet (UV) light at a wavelength of 254 nm has been used in Japan to detect latentˆngerprints. However, the degradation of DNA at 254 nm UV has become a problem for short tandem repeat (STR) typing. In this study, 306 nm UV light that gives a relatively sharpˆngerprint image was selected as a new light source for our imaging instrument, and its eŠect on STR typing was compared to that of the 254 nm UV light. Twenty microliters of saliva and 3 ml of blood were dried on glass slides and irradiated from 5 20 cm with 306 nm or 254 nm UV light for 0.5 30 min. DNA extracted from the UV-irradiated samples was quantiˆed using real-time PCR assay and STR typing was performed. Both wavelengths reduced the amounts of DNA, as irradiated at the shorter distance or for the longer time. However, the reduction of the amount of DNA was less under the 306 nm UV light than the 254 nm. When the 306 nm UV light was irradiated from 5 cm, full STR proˆles were obtained from the saliva and blood samples within 10 and 3 min, respectively. In addition, when it was irradiated from 20 cm, full STR proˆles were obtained from all the samples in spite of irradiation for 30 min. It was indicated that the 306 nm UV light could be useful as a new light source. However, the use of the 306 nm UV light at a short distance or for a long time should be avoided as much as possible for STR typing.

To obtain DNA typing results from forensic biological samples is occasionally di‹cult, since they are complex and diverse. AmpF STRIdentiˆlerPlus PCR Ampliˆcation Kit (IDPlus) was developed to improve the success rate of DNA typing from such samples by Applied Biosystems. Forensic biologists must be cautious in DNA typing when considering the characteristics of the Kit used for analyzing in such samples. Therefore, to reveal to the characteristics of IDPlus Kit is very important to increase the reliability and probative value of DNA typing in the trial and investigation. In this study, IDPlus is evaluated for the characteristics and the DNA detectability. We also compared IDPlus with AmpF STRIdentiˆlerPCR Ampliˆcation Kit (ID) by analyzing samples di‹cult to obtain DNA types. The results from the PCR inhibitor study using bloodstain on soil or denim indicated that IDPlus was more resistant to PCR inhibitors than ID. In the study of analyzing degraded DNA, there were no signiˆcant diŠerences to be found except for the sensitivity. The peak corresponding to insu‹ciently ampliˆed products due to a point mutation on primer binding site was higher using IDPlus than ID in electropherogram. Also the anomalous peaks which were observed in analyzing same mammal animal DNA samples were higher. Hence, IDPlus is considered to be very eŠective leverage to samples analysis which is di‹cult to detect allele due to PCR inhibitors. The primer binding ability to the template DNA was altered when using IDPlus compared with the ID so this should be taken into consideration. Forensic

A validation study was performed on the new STR (Short tandem repeat) multiplex PCR Kit for human identiˆcation: AmpF STRIdentiˆlerPlus PCR Ampliˆcation Kit (IDPlus) released from Applied Biosystems. IDPlus contains the sameˆfteen loci as AmpF STRIdentiˆlerPCR Ampliˆcation Kit (ID) which is currently widely utilized in forensic DNA analysis. Consequently, IDPlus has the same discrimination ability as ID. According to the manufacturer, this kit has higher sensitivity, more resistant to PCR inhibitors and less background noise in electrophoresis. Thus, IDPlus was expected to be applied to forensic samples di‹cult to DNA proˆling using ID. An applicability of IDPlus to forensic STR analysis was evaluated. Our study conˆrmed that IDPlus was more sensitive than ID. IDPlus has about 1.4 times to 1.5 times higher peak than ID when using the same PCR cycle number (28) for both kits. While ID has only one PCR protocol, IDPlus has two PCR protocols diŠering in cycle number: 28 and 29. To clarify the basic ability we compared heterozygous peak height ratio, stutter, intra-color balance, inter-locus balance among IDPlus to both protocols and ID from 94 individual DNA samples. Lower peak height ratio and the larger standard deviation in heterozygous samples were observed when using IDPlus 29 cycle compared to IDPlus 28 cycle. There was no signiˆcant diŠerence between ID and IDPlus 28 cycle and between ID and IDPlus 29 cycle about the peak height ratio. Stutter ratios signiˆcantly were diŠer-ent in some loci between ID and IDPlus. Although Applied Biosystems supplies one stutter ratio toˆlter peaks for each locus to IDPlus in spite of having two PCR protocols, it was conˆrmed that the same stutter ratio could properˆlter out stutter peaks for both cycle numbers. With regard to intra-color balance and inter-locus balance, IDPlus 28 cycle tended to be the highest in balance and ID tended to be the

A quantitative PCR procedure using a newly designed primer set for ampliˆcation of human speciˆc DNA sequence D17Z1 for quantiˆcation of human DNA has been developed. The procedure was compared with the existing quantiˆcation method, UV absorption method and slot blot hybridization method. The lower limit of quantiˆcation, the reproducibility of results, quantiˆcation of degraded DNA, quantiˆcation speciˆcity and application for forensic casework samples were examined using each technique.The quantitative PCR method consumed the least amount of DNA and could quantify as little as 0.0015 ng of DNA, and its reproducibility was comparable with the UV absorption method. The STR typing of degraded DNA samples quantiˆed by slot blot hybridization method and quantitative PCR method were properly detected and typed. Since the bacterial DNA samples were not detected and quantiˆed by slot blot hybridization method and quantitative PCR method, they are useful for human-speciˆc DNA quantiˆcation of forensic evidence samples containing bacterial DNA. In the experimental application of the quantitative PCR method for various forensic casework samples, almost every STR loci were properly detected and it demonstrated a beneˆcial eŠect for actual forensic identiˆcation analysis. Furthermore, the quantitative PCR method was also useful in determining the proper amount of template DNA extracted from the sample suspected to include ampliˆcation inhibitor. In the results of the comparisons, the human speciˆc DNA quantiˆcation using the technique of quantitative PCR is more practical for forensic DNA analysis than the other methods.