Screening of folate status with use of dried blood spots on filter paper.
(1/32)
BACKGROUND: Dried blood spots (DBS) on filter paper have been a successful and economical matrix for neonatal screening. OBJECTIVE: Our objective was to develop and evaluate an optimized method for DBS folate analysis and to assess DBS folate stability. DESIGN: DBS were eluted from paper by sonication in 5 g ascorbic acid/L containing 0.1% (by vol) Triton X-100 and hemoglobin folate values (HF; as pmol/g) were calculated from DBS eluate folate and hemoglobin concentrations. RESULTS: Over 95% of DBS folate was eluted during a standardized sonication cycle and DBS folate assay reproducibility was acceptable both within (CV: <8%) and between (CV: <9%) runs. HF means (+/-1 SD) from finger-stick DBS and conventional venous methods were 2513 +/- 1144 and 2607 +/- 1195 pmol/g, respectively, in blood samples taken concurrently from 80 donors, and they correlated well (r = 0.97, P < 0.001). HF values and erythrocyte folate measures may be interconverted by using the mean cell hemoglobin concentration. CONCLUSION: The DBS matrix has potential as an inexpensive and practical option for folate screening studies. (+info)
Streptokinase versus alteplase and other treatments for acute and delayed thrombolysis of blood stains in clothing.
(2/32)
OBJECTIVE: To assess the usefulness of heparin, alteplase, and streptokinase in removing blood stains. DESIGN: Randomised controlled trial. SETTING: Hospital laundry. INTERVENTIONS: Blood stains were allocated to treatment with alteplase, streptokinase, heparin, a commercial enzymatic stain remover, or no treatment at all after three or seven hours and then washed in hot or cold water two hours later. RESULTS: Both hot water and early treatment were strongly associated with improved stain removal. All four treatments were associated with a worse outcome than no treatment at all, although for streptokinase this trend did not reach significance. The commercial stain remover gave the worst results of all treatments tested. CONCLUSIONS: Contrary to popular wisdom, hot water is much more effective than cold in removing blood stains. Methodologically rigorous research and evidence based principles are needed within the laundry industry, and the role of thrombolytic drugs should be assessed further. (+info)
Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.
(3/32)
AIM: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA). METHODS: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method. RESULTS: All 144 pairwise comparisons of HV1 and HV2 mtDNA fragments were successfully resolved by the DHPLC method. Forensic proficiency test standards were successfully resolved and DHPLC match/non-match results agreed with sequencing results provided by the test providers. The DHPLC method successfully identified one questioned sample that was prepared by the test provider as a body fluid mixture. MtDNA amplicon mixtures could be separated into their constitutive components by DHPLC and fraction collection approaches. CONCLUSIONS: DHPLC methods provide the forensic scientist with a powerful tool to rapidly screen mtDNA and may result in standardized methods to resolve mtDNA mixtures. These advances will allow mtDNA analysis in cases not previously examined by current sequencing-based approaches and could allow more forensic case samples to be entered into the proposed mtDNA Combined DNA Index System (CODIS trade mark ) databank as a result of mtDNA mixture resolution. (+info)
Identification of human remains by immobilized sequence-specific oligonucleotide probe analysis of mtDNA hypervariable regions I and II.
(4/32)
AIM: A rapid analysis of mitochondrial DNA (mtDNA) sequences with an array of immobilized sequence-specific oligonucleotide (SSO) probes was tested on 18 skeletal elements recovered from mass graves in Croatia, which could not be genotyped with common forensic nuclear DNA systems (PM+DQA1 and short tandem repeat analysis). METHODS: We used duplex polymerase chain reaction (PCR) amplification of the mtDNA hypervariable regions I and II (HVI and HVII) (444 bp and 415 bp amplicons, respectively) and subsequent linear array typing, which targets six polymorphic regions and two additional sites within the human mtDNA HVI and HVII. The remaining amplified products were subjected to direct sequence analysis to obtain complete sequence information for the targeted HV regions. RESULT: Duplex PCR amplification of the mtDNA HVI and HVII was successful in providing sufficient product for typing with the array of SSO probes in 14 out of the 18 sample extracts. We report here the sequence match of one set of remains with a panel of immobilized SSO probes, followed by direct sequence analysis. The corresponding mtDNA haplotype obtained for the bone sample and the putative maternal reference was unique in a database of 105 randomly selected Croatian individuals. CONCLUSION: Mitochondrial DNA typing with an array of immobilized SSO probes can be a benefit to forensic DNA analysis of mass disaster remains and identity testing of single and mass graves. (+info)
A rapid chemiluminescent method for quantitation of human DNA.
(5/32)
A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples. (+info)
Influence of template DNA degradation on the genotyping of SNPs and STR polymorphisms from forensic materials by PCR.
(6/32)
Detection of single nucleotide polymorphisms (SNPs) and short tandem repeat (STR) polymorphisms by PCR is widely used to analyze degraded DNAs in forensic science. The success of DNA analysis from human remains largely depends on the quality of the template DNA. We examined two SNPs (HLA-DQA1 and ABO) and two STR polymorphisms (VWA and CD4) by SSCP gel or denaturing gel electrophoresis, using two kinds of degraded DNA samples (165 teeth and blood stains contaminated with saliva) derived from the same person and investigated the influence of template DNA degradation on genotyping. As the degradation of DNA proceeds, unbalanced amplification of alleles occurred in the analysis of both SNPs and STRs, followed by allele drop, and further by loss of amplification. Non-target allelic products of STRs were amplified from highly degraded DNA samples; however, false allelic products of SNPs were not amplified from them. Amplification efficiency increased in proportion to the decrease of PCR target size, but reduction of the PCR target sizes also increased the chances of amplifying contaminating DNA, especially in highly degraded DNA specimens. The present results will help investigators to evaluate the genotyping of highly degraded DNA samples in forensic casework. (+info)
A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples.
(7/32)
We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine. (+info)
Polymorphism of LPL Locus in Japanese and comparison of PCR amplification efficiency from degraded DNA between LPL locus and the D21S11.
(8/32)
The short tandem repeat (STR) polymorphism of the lipoprotein lipase (LPL) locus was amplified by PCR and analyzed using denaturing polyacrylamide gel electrophoresis followed by silver staining. Among 158 DNA samples from the Japanese population, six alleles were observed. When the sequences of the allelic products were compared, each allelic segment contained 7 and 9-13 TTTA tetranucleotide repeat motifs. Genotypic distribution met Hardy-Weinberg expectations, and included heterozygosity was 48.8%. Most of the Japanese genotypes allele 10. When PCR amplification efficiency for the LPL locus from degraded DNA was compared with that for the D21S11 locus in terms of amplification size, increase in amplification size showed a considerable influence on amplification efficiency, producing inaccurate amplification, such as unbalanced amplification, or amplification of non-target PCR products. These results suggest that reduction in amplification size increases the accuracy and efficiency of PCR amplification from highly degraded DNA. (+info)