DNA profiling of bloodstains on linen pretreated with remedies used for cleaning and maintaining clothes

Summary Linen was pretreated with 20 remedies for cleaning and maintaining clothes and 20 l blood was applied on each sample and dried. Restriction enzyme digest of bloodstain DNA was irregularly inhibited by highly concentrated residues of 2 detergents and a stainremover and colour-brightener. An additional dialysis step to purify DNA (Gill 1987) reliably prevented disturbance. High molecular DNA was obtained in every case and bandshifts were not observed.     

Extraction of high quality DNA from bloodstains using diatoms

A simple method is described for the extraction of high quality DNA for PCR amplification. The DNA was extracted by using Chelex-100 ion exchange resin or a special cell lysis buffer containing proteinase K. For further purification the DNA was bound to silica in the presence of a chaotrophic agent. Hence it is possible to unlimitedly wash the bound DNA and inhibitory substances are removed. By using diatoms as a source of silicates, this method is very economical and can therefore be used as a routine method.     

Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains

Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5′ defined sequence and alter the number of bases on the 3′ end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.     

Extraction strategy for obtaining DNA from bloodstains for PCR amplification and typing of the HLA-DQa gene

Summary A simple, practical approach for the extraction of PCR-amplifiable DNA for the HLA-DQa gene from bloodstains deposited on various substrates is described. DNA from bloodstains is purified using Chelex 100 ion-exchange resin and then amplified. If amplification is not achieved, the extract is washed through a Centricon 100 dialysis/concentration tube. If the second amplification of this extract produces a negative result, the extract is processed with Chelex 100 again. This approach has been found to be reliable, safe, efficient and economical.     

GEDNAP IV and V. The 4th and 5th stain blind trials using DNA technology

In the collaborative exercise GEDNAP IV one EDTA blood sample (2 ml) and 5 bloodstains (0.5 ml on cotton) were investigated and in GEDNAP V, a total of 8 bloodstains (0.5 m1 on cotton), including 2 mixed bloodstains. DNA typing was carried out using the RFLP systems YNH24/Hinf I and MS43a/Hinf I and the PCR systems HLA DQ, D1S80, ApoB and YNZ22. In both exercises approximately 20 laboratories obtained results using the RFLP systems. Of the PCR systems, DIS80 was the most commonly used (14 labs in GEDNAP IV; 18 labs in GEDNAP V). The interlaboratory standard deviation for YNH24 in both exercises was approx. 0.6%, for MS43a 0.7–2.2% (GEDNAP IV) and 0.4–1.4% (GEDNAP V), depending on the fragment size. The fragment size calculation performed in each laboratory yielded a standard deviation twice that obtained when the fragment size calculation was performed centrally (IfR, Münster). In GEDNAP III, a system-specific corridor was developed to define the limits of deviation; this was modified for the present study by combining the fragment size ranges of YNH24 and MS43a. In both studies a subgroup of laboratories was involved in preliminary exercises using three PCR VNTRs and the system HLA DQ. Owing to the substantial variation in experience of the participating laboratories with PCR typing the results obtained in these two studies do not fulfil the basic quality criteria of the GEDNAP studies.     

Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins

Summary Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), ₂-macroglobulin (₂-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human ₂-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next.Supported in part by the Cooperation Research Program of the Primate Research Institute     

Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues

Summary DNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at –25°C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day post mortem. However, more than 90% of DNA samples up to at least 3 days post mortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed good RFLP patterns. However, no obvious relation between qualities of DNA analyzed by detection of RFLP and quantities of total and high-MW DNA became apparent. A case of familial relationship was ascertained by DNA fingerprints. Since DNA of good quality can be recovered from muscle tissues in large quantities, DNA extraction from muscle tissues and detection of RFLP patterns should be very useful for individual identification in autopsy cases.     

The effect of bleaching agents on the DNA analysis of bloodstains on different floor coverings

International Journal of Legal Medicine - Blood at crime scenes is one of the most significant traces of evidence in investigation proceedings. Cleaning up these traces with household cleaning...     

Methods enhancement for improved recovery of human DNA from forensic blood samples on different fabrics using the DNA IQ System

DNA typing of biological samples can be a challenging task due to the presence of small amounts of DNA and/or a high content of PCR inhibitors. Our aim is to develop a protocol to recover DNA suitable for DNA typing from blood-stained fabrics based on the DNA IQ System. Blood stains on different fabrics were buried in different types of soil and left for 1–7 days. The samples were then recovered and DNA was extracted with a modified DNA IQ System protocol, involving a modification in the preparation of the Wash Buffer. The samples extracted with the modified protocol showed a higher amount of recovered DNA compared with the recommended protocol. The removal of isopropanol from the Wash Buffer composition contributes to a higher amount of recovered DNA.     

Bloodstain examination and DNA typing from hand-washed bloodstains on clothes

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.     

PCR-based typing of DNA extracted from cigarette butts

Summary Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately < 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.     

Touch DNA: The effect of the deposition pressure on the quality of latent fingermarks and STR profiles

Latent fingermarks (FMs) present unique, and sometimes the only, evidence found at a crime scene. Several factors affect their quality, including deposition pressure (DP). Its effect on FM size and quality, and on STR amplification success rate, is an emerging area of interest in forensic science. This study examined 540 FM samples, each consisting of index, middle and ring fingers, deposited by 30 donors on glass, polythene (PE) and paper under a range of weights from 0.1 to 10 kg. Both length and width of FMs increased with the increasing DP. FMs deposited under lower (≤0.5 kg) DPs varied in size (p < 0.01), while those deposited at higher (≥3 kg) DPs were more consistent. FM quality on glass and PE, as determined by the AFIS minutiae count and by a fingerprint examiner on a scale from 0 to 4, improved with the increasing DP, but it deteriorated on PE at DP of 10 kg. FM quality on paper continued to improve from DP of 1 kg up to the maximum DP of 10 kg. The effect DP has on the efficacy of DNA profiling from latent FMs was significant as shown by an increase in the DNA amount recovered, the number of amplified loci per FM sample, and the number of forensically useful DNA profiles (defined here as those with ≥8 full STR loci detected) as DP increased. This effect was most pronounced with PE (R = 0.98) and paper (R = 0.96). Altogether, the success rate of DNA profiling varied from 16.3% in FMs deposited on paper to 21.2% and 22.5% of those on PE and glass. The highest number of useful DNA profiles was obtained from glass under DP of 10 kg. Forensically useful FMs obtained at low (≤1 kg) DP from all three substrates significantly outnumbered that of STR profiles, while an opposite, though less pronounced trend, was observed at high (≥3 kg) DP on PE and paper. Application of the simple device for collecting of FMs under controlled pressure designed for this study, and the palm-up mode of FM deposition as described, allowed us to eliminate the undesirable effect of the hand self-weight and to objectively assess the actual effect of increasing DP on FM size and quality, as well as on the efficacy of DNA profiling.     

Influence of soil storage and exposure period on DNA recovery from teeth

A study was performed to determine the influence of garden soil on the deoxyribonucleic acid (DNA) recovery from teeth depending on the duration of storage. In the first series 24 teeth supplied by dentists were exposed to garden soil storage for a maximum of 18 weeks. Selected samples were excavated for DNA extraction at time intervals of 6,12 and 18 weeks. For the second series 20 teeth were stored for one year in garden soil. Following phenol/chloroform extraction with decalcification (first series) and without decalcification prior to extraction (second series) DNA was quantified, amplified using the polymerase chain reaction (PCR) for the tandem repeat loci D1S80, tyrosine hydroxylase, intron 1 (TH01) and Von Willebrand factor, intron A (VWA) (first series), human alpha fibrinogen (FGA) (second series) and sequenced in the hypervariable regions 1 and 2 (HV1, HV2) of the mitochondrial DNA (second series). The DNA concentration of the extracts after the first 6 weeks in soil was reduced by more than 90%. Amplification and direct sequencing of HV1 and HV2 of the mitochondrial genome was the most successful DNA technique. Received: 5 June 1998 / Received in revised form: 11 July 1998     

An evaluation of the effect of microwave irradiation on bone decalcification aimed to DNA extraction

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen–antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction.     

DNA extraction from mixtures of body fluid using mild preferential lysis

Summary A modification to the DNA extraction method preferential lysis (Gill et al. 1985) is proposed which can be applied to DNA mixtures of vaginal cells and spermatozoa. In mixtures with a low sperm content the further loss of sperm DNA caused by the extraction can be avoided by using mild preferential lysis. Amplification by PCR (polymerase chain reaction) then yields sufficient DNA to be able to identify both components in the mixture.     

PDGF-AB对成纤维细胞增殖及DNA合成的影响

目的：探讨ＰＤＧＦ－ ＡＢ促进伤口愈合及其在瘢痕增生中的作用机理。方法：取体外培养的人正常皮肤及增生性瘢痕成纤维细胞（ｎｏｒｍａｌｓｋｉｎｆｉｂｒｏｂｌａｓｔ,ＮｓＦｂ,ｈｙｐｅｒｔｒｏｐｈｉｃ ｓｃａｒｆｉｂｒｏｂｌａｓｔ,ＨＴｓＦｂ） ,用ＭＴＴ和３Ｈ－ ＴｄＲ掺入法观察ＰＤＧＦ－ ＡＢ对两种细胞增殖及ＤＮＡ合成的影响。结果：ＰＤＧＦ－ ＡＢ对两种成纤维细胞增殖及ＤＮＡ合成均有明显刺激作用,且均呈剂量依赖性关系,但作用有差异。结论：ＰＤＧＦ－ ＡＢ可能通过刺激成纤维细胞增殖及ＤＮＡ合成促进伤口愈合,但同时在瘢痕增生性疾病中可能起重要促进作用。     

Separation of Y-chromosomal haplotypes from male DNA mixtures via multiplex haplotype-specific extraction

In forensic analysis, the interpretation of DNA mixtures is the subject of ongoing debate and requires expertise knowledge. Haplotype-specific extraction (HSE) is an alternative method that enables the separation of large chromosome fragments or haplotypes by using magnetic beads in conjunction with allele-specific probes. HSE thus allows physical separation of the components of a DNA mixture. Here, we present the first multiplex HSE separation of a Y-chromosomal haplotype consisting of six Yfiler short tandem repeat markers from a mixture of male DNA.     

Comparison of DNA extraction and amplification from ancient human bone and mummified soft tissue

South american precolumbian male mummies were employed as source material for a comparative investigation of bone and soft tissues by DNA analysis. The suitability of the DNA extracts from both sources was tested and evaluated by their effectiveness as target DNA in PCR amplifications. The results suggest that skeletal material should be given preference over soft tissues for PCR analysis if the material is severely degraded. This seems to be independent of the specific anatomical origin of the samples.     

The origin of unknown source DNA from touched objects

The presence of DNA in a criminal investigation often requires scrutiny in relation to how it came to be where it was found. There is a paucity of data with respect to the extent to which one can assume that the last person handling an object, which has previously been touched by others, will contribute to the DNA profile generated from it. There are limited data in detailing the extent to which any foreign DNA is picked-up from a previously touched object and transferred to subsequently touched objects. This study focuses on DNA transfer and persistence on a knife handle after multiple handlings with the knife by different individuals soon after each other, as well as handprints left on flat DNA-free surfaces immediately after touching a knife handle with a known history of prior handling.The profiles of later handlers of a knife are more prominent than earlier handlers; however, the last handler is not always the major contributor to the profile. Proportional contributions to the profiles retrieved from knife handles vary depending on the individuals touching the knife handle. They can also vary when knife handles have been handled in the same manner by the same individuals in the same sequence on different occasions. Hands readily pickup DNA left on objects by others and transfer it to subsequently touched objects. The quantity of foreign DNA picked up by a hand and deposited on subsequently touched objects diminishes as more DNA-free objects are handled soon after each other.Caution is advised when considering how DNA from different individuals may have been transferred to the object from which it was collected.     

DNA within cars: prevalence of DNA from driver,passenger and others on steering wheels

ABSTRACT

Cars are frequently involved in criminal activities and sampled for DNA to assist investigations. To improve our awareness of DNA transfer, persistence, prevalence and recovery (DNA-TPPR) within cars we studied DNA profiles from samples collected from several sites in the front compartment of cars with known histories and occupancies. Here findings relating to steering wheels are reported. Each of the four quarters of the rim as well as the centre column, of four cars, provided good quantities of DNA for profiling. The driver was observed as the sole, major or co-major in 19/20 profiles, and as a minor in the remaining profile generated from these samples. Known close associates, including co-resident partners and passengers/friends, as well as other unknown individuals, who had not driven the car, are also detected on many of the sampled steering wheel sites. More studies are required to improve our awareness of DNA-TPPR and to generate data to help determine probabilities for different profile types and levels of specific contributions given specific circumstances relating to steering wheels, as well as several other relevant areas within cars, to assist sample targeting and activity level assessments.