Distribution of the AMPFLPs YNZ22, 3′APOB and COL2A1 in the population of Galicia (NW Spain)

Two different electrophoretic methods were used for typing three amplified fragment length polymorphisms (AMPFLPs), (3′ApoB, YNZ22 and COL2A1) in a Galician (NW Spain) population sample. Because of the problems of anomalous mobility for the 3′ApoB system and the intermediate alleles found in the COL2A1 system, the use of automated sequencers and denaturing conditions is recommended for typing these two systems. Nevertheless, simple electrophoretic methods, such as the PhastSystem, can be used for YNZ22 typing. Although intermediate COL2A1 alleles can be distinguished with the sequencers, a binning approach was adopted for comparison purposes. The population sampled was in Hardy-Weinberg equilibrium for the three systems using an exact test. This type of statistical analysis is more appropriate when the number of alleles in a system is high. No significant differences with other Caucasian populations were found for the three systems studied. The characteristics of the polymorphisms, shown by 3′ApoB, YNZ22 and COL2A1, reflected in the statistical parameters studied, demonstrate that these AMPFLPs are of considerable interest for forensic purposes.     

Study of three AMPFLPs (D1S80, 3′ApoB and YNZ22) in the population of the North of Portugal

Allele and genotype frequencies for D1S80, 3′ApoB and YNZ22 loci have been determined in a population sample of the North of Portugal using the polymerase chain reaction (PCR) amplification and nonradioactive detection. The distribution of genotypes in the three polymorphisms studied is in agreement with expected values according to the Hardy-Weinberg equilibrium. The combined chance of exclusion for the three systems is 0.96, and the combined power of discrimination is 0.99.     

Validation and casework application of a Y chromosome specific STR multiplex

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor’s alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.     

The esterase D polymorphism as revealed by isoelectric focusing in ultra-thin polyacrylamide gels

The polymorphism of human red cell esterase D (EsD) was studied using isoelectric focusing (pH 4-6) in ultra-thin polyacrylamide gels. Typing was possible without the EsD isozymes attaining true equilibrium focusing conditions. Using this single method, six phenotypes (EsD 1, 2-1, 2, 5-1, 5-2 and 5) could be recognized in the White population of south-east England. Family studies showed these to be controlled by three co-dominant alleles and the gene frequencies were calculated to be EsD1 0.8856; EsD2 0.0946 and EsD5 0.0198. For successful and reliable EsD typing by this method, the electrophoretic system must be carefully optimized with respect to the duration of electrophoresis and the temperature attained in the gel during the electrophoretic run.     

VNTR遗传标记的亲权鉴定研究

采用Amp－FLP技术研究人类血液、组织VNTR位点D1S80（pMCT118）、D17S30（pCNZ－22）和ApoB3’位点的遗传多态性，并应用于亲权鉴定案件，获得满意结果。D1S80、D17S30和ApoB3’的DP值分别为0．962、0．956和0．960，累积父权排除率（EPP）为94．51％，远远高于传统血型的DP值和EPP值，是亲权鉴定和个体识别有效方法。     

Validation and casework application of a Y chromosome specific STR multiplex

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor's alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.     

A report of the 2002–2008 paternity testing workshops of the English speaking working group of the International Society for Forensic Genetics

The English Speaking Working Group (ESWG) of the International Society for Forensic Genetics (ISFG) offers an annual Paternity Testing Workshop open to all members of the group. Blood samples, a questionnaire and a paper challenge are sent to the participants. Here, we present the results of the 2002–2008 Paternity Testing Workshops with the objective to evaluate the uniformity of DNA-profiling and conclusions of the participating laboratories as well as to clarify tendencies in typing strategies and biostatistical evaluations of the laboratories. The numbers of participating laboratories increased from 46 in 2002 to 68 in 2008. The results showed an increasing degree of concordance concerning methods and DNA systems used and a high degree of uniformity in typing results with discrepancies in 0.1 and 0.3 % of all submitted PCR-based results. The paper challenges showed uniformity in the calculation of the weight of evidence for simple cases with straight-forward genetic constellations. However, a high degree of variation existed in complex scenarios with rare genetic constellations such as genetic inconsistencies/possible silent alleles, rare alleles and haplotypes.     

6个miniSTR基因座荧光标记复合扩增的遗传多态性

目的评价6个miniSTR基因座在DNA高度降解检材中的法医学应用价值,并调查广东汉族人群6个miniSTR基因座的遗传多态性。方法采用两个复合扩增PCR体系、四色荧光标记及毛细管电泳技术,对D1S1677,D2S441,D4S2364,D10S1248,D14S1434,D22S1045基因座进行基因型检测。结果6个miniSTR基因座均获得了清晰的基因型分型结果,扩增片段均小于120bp,分别检出7、7、5、8、8、7个等位基因和14、11、11、19、12、14种基因型,基因型分布均符合Hardy-Weinberg平衡。6个miniSTR基因座在广东汉族群体的个人识别率和非父排除率分别依次为0.863、0.895、0.792、0.894、0.814、0.904和0.392、0.360、0.353、0.568、0.378、0.513。10例IdentifilerTM试剂盒未能正确分型的高度降解DNA样本,采用6个miniSTR基因座复合扩增体系检测均提高了分型成功率结论6个miniSTR基因座荧光标记复合扩增体系在DNA高度降解检材的检测中具有较高的应用价值,并且在广东地区汉族群体中具有较好的遗传多态性。     

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Several laboratories have reported the occurrence of a split or n − 1 peak at the vWA locus in PowerPlex^® 16 and PowerPlex^® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n − 10/n − 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n − 10/n − 18 artifact in PowerPlex^® 16 and PowerPlex^® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex^® 16, PowerPlex^® Y, PowerPlex^® ES, AmpFlSTR^® Profiler Plus^® ID, AmpFlSTR^® Cofiler^®, and AmpFlSTR^® SGM Plus^® kits.     

Possibilities for using bedside cards as secondary comparison in trace element studies with the PCR technique]

When fresh blood is not available as a control in stain investigations extracted teeth, hair, preserved tissue samples, histological slides, cigarette butts or used stamps can also be used. This paper reports on a stain investigation performed 7 months after the death of the victim, where a bedside card from the medical records was successfully employed as a control blood sample. In a series of 10 bedside cards up to 9 years old, the investigation with the PCR method showed recognizable patterns in the STR systems SE 33 and TC 11. Matching patterns could be found from the 4 sections of each card (anti A, anti B, anti AB and anti D). A comparison of the oldest card with a fresh blood sample of the patient also showed matching patterns. AMPFLPs were successful with more recent cards. Using the PCR method typing of bedside cards from medical records up to 10 years old can be used in stain investigations.     

Minisequencing-based genotyping of Duffy and ABO blood groups for forensic purposes

Duffy and ABO blood group genetic polymorphisms were studied by minisequencing analysis of single-nucleotide polymorphisms (SNPs) at nucleotide positions--33, 125, 265, and 298 of the Duffy gene and at nucleotide positions-261, 297, 467, 646, and 703 of the ABO gene. In an Italian population sample, we found four alleles and seven genotypes for the Duffy and six alleles and 16 genotypes for the ABO systems. The lower limit for reproducible results was 200 pg DNA, with a range of up to 10 ng and an optimum at 1 ng. All of the 16 analyzed inclusive paternity tests were also consistent with parentage and two out of four inconsistencies with parentage cases were excluded by one or more SNPs. Although Duffy and ABO SNP typing show lower informativeness than most current forensic tests, their robustness, the limited population distribution of FY* Fy type, and the sensitivity of the minisequencing technology suggest that these markers can be useful in selected forensic applications.     

Additional sequence characterization of NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.     

Accurate STR allele designations at the FGA and vWA loci despite primer site polymorphisms

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.     

An agarose gel electrophoretic method for typing phosphoglucomutase-1, esterase D, or glyoxalase I

A conventional agarose gel electrophoretic method was described for typing phosphoglucomutase-1, esterase D, or glyoxalase I as single systems. Bloodstain extracts were absorbed into 1-mm-thick agarose gels via an application mask. The electrode wick distance was 12 cm and electrophoresis was carried out at 400 V at 6 degrees C. The electrophoretic run times were 30 min for glyoxalase and 1 h for esterase D or phosphoglucomutase. This method is reliable and produces highly resolved band patterns. Additionally, the shorter separation times as a result of the increased voltage gradient permitted typing of more samples in a given time period compared with presently used methods. This technique requires little technical expertise and can be incorporated into the laboratory at a minimal cost.     

Allele frequencies of six miniSTR loci in the population of Northern Portugal

A possible approach to try to recover information from degraded DNA is to reduce the size of the PCR products by designing primers that bind as close as possible to the STR repeat region, known as miniSTRs. Allele frequencies and forensic parameters for the six miniSTRs loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 were investigated in a sample group consisting of 228 anonymous apparently healthy unrelated individuals living in North of Portugal. The results show that all loci were in Hardy–Weinberg equilibrium. The combined power of discrimination and power of exclusion for the six loci were 0.99999 and 0.9789, respectively. All but one (D4S2364) loci showed a moderate degree of polymorphism (observed heterozygosity >0.6). The allele sizes ranged between 66 and 118 bp in our population, which is beneficial for typing degraded samples than those of a commercial STR kit.     

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.     

A repository of 14 PCR-loci Italian gene frequencies in the World Wide Web

A collection of 6830 typing results produced by the Immunohematology Laboratory at the UCSC, pertaining to 11 STRs (FES/FPS, vWA31, HUMTH01, F13A1, MBP, D21S11, D7S460, D18S51, CD4, TPOX, CSF1PO) and 3 AmpFLPs (D1S80, APO-B, COL2A1), is publicly available as an electronic archive at a website.     

Comparison of German population data on the apoB-HVR locus with other Caucasian, Asian and Black populations

A population study of 505 unrelated individuals from Southwestern Germany was carried out on the 3′-apoB hypervariable region (HVR). After amplification via polymerase chain reaction (PCR) and agarose gel electrophoresis, 15 different alleles and 47 genotypes were observed. The most common alleles were hypervariable elements (HVE) 37 and 35 with an allele frequency of 0.374 and 0.244, respectively. The heterozygosity index was calculated to be 78.4%. Allele frequencies of this study are compared with results from other databases obtained from a French, a Spanish, an Asian and an American (Black) population.     

Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC) and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58 and 0.322, respectively. For the H19FR2, two haplotypes and three genotyes were observed, and the Dp, PIC and PE were 0.626, 0.37 and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population, and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles.     

Determination of phenotypes of salivary amylase in liquid saliva and saliva stains

An isoelectric focusing method is described for typing salivary amylase in liquid saliva and saliva stains. The estimated gene frequencies in a British population, calculated on the basis of three alleles operating at a single locus, were Amy 1, 0.909; Amy 2, 0.065; Amy 3, 0.026. This system may be useful in forensic investigations.