Marie Allen

Mitochondrial DNA Sequencing of Shed Hairs and Saliva on Robbery Caps: Sensitivity and Matching Probabilities

Sequencing of mitochondrial DNA (mtDNA) has been used for human identification based on teeth and skeletal remains. Here, we describe an amplification system for the mtDNA control region (D-loop) suited for the analysis of shed hair, which constitutes the most common biological evidence material in forensic investigations. The success rate was over 90% when applied to evidence materials such as shed hair, saliva stains and saliva on stamps. The analysis of evidence materials collected from three similar robberies revealed the presence of mtDNA sequences identical to those of the suspects in the three crimes. The use of mtDNA control region sequences for individual identification was evaluated. The probability of identity by chance for the mtDNA types of the suspects in the robberies was found to vary between Pr = 0.017 - < 0.0017, depending on the reference population used, emphasizing the need for large population databases to obtain the appropriate estimate.

Association of susceptibility to multiple sclerosis in Sweden with HLA class II DRB1 and DQB1 alleles

The association of MS with HLA class II alleles was studied by PCR-based typing of the DQA1, DQB1, DRB1, and DPB1 loci in 94 Swedish patients with relapses and remissions of the disease. The haplotype DRB1*1501-DQA1*0102-DQB1*0602 was found to be positively associated and three haplotypes were found to be negatively associated with MS. Linkage disequilibrium makes it difficult to assess whether DRB1 or DQB1 plays the primary role in the disease association, while the association with DPB1 and DQA1 appears to be secondary to that of DQB1 and DRB1. Two of the three haplotypes negatively associated with MS carry the DQB1*0301 allele. Also, the negatively associated DRB1*0401-DQA1*0301-DQB1*0301 haplotype differs from those with nonassociated DRB1*0401-DQA1*0301-DQB1*0302 haplotype only at DQB1. These results suggest that DQB1 alleles, as well as some DRB1 alleles, are involved in susceptibility and protection to MS. In searching for sequence motifs in the DR beta chain associated with MS susceptibility, all DRB1 alleles on haplotypes positively associated with MS, including the DRB1*1501, were found to encode a Val at position 86 of the DR beta chain. Also, DRB1 alleles that are negatively associated with MS all encode a Gly at position 86, suggesting that the residue at position 86 may be critical in conferring susceptibility and protection to MS. Finally, when the effect of the DRB1*1501 haplotype was removed there was no support for the hypothesis that MS is associated with a putative DQ-alpha beta heterodimer, encoded for by certain DQA1 and DQB1 alleles.

Origin of the U87MG glioma cell line: Good news and bad news

The DNA profile of the publicly available and widely used glioma cell line U87MG is quite different from that of the original cell line. U87MG: Not what it used to be A cell line commonly used for research on gliomas is found to be different from the original tumor from which it was derived. The authors, whose laboratory developed the cell line almost 50 years ago, compared the genetics of this line (obtained from ATCC) with those of the original tumor. They report that the DNA profile of the current cell line differs from that of the original cells but that it is likely to be a human glioblastoma cell line with unknown origins. This misidentification of a widely studied cell line reinforces the need for researchers to carefully validate the cell lines used in their research. Human tumor–derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

Sequencing of in vitro amplified DNA

Publisher Summary The polymerase chain reaction (PCR) is based on the use of two oligonucleotides to prime DNA polymerase-catalyzed synthesis from opposite strands across a region flanked by the priming sites of the two oligonucleotides. By repeated cycles of DNA denaturation, annealing of oligonucleotide primers, and primer extension, an exponential increase in copy number of a discrete DNA fragment is achieved. This chapter reviews alternate methods for the generation of sequencing templates from amplified DNA and sequencing. Templates for DNA sequencing have been generated by inserting the target DNA into bacterial or viral vectors for multiplication of the inserts in bacterial host cells. By using PCR, templates for sequencing can be generated more efficiently than with cell-dependent methods either from genomic targets or from DNA inserts cloned into vectors. Sequencing the PCR products directly has two advantages over sequencing of cloned PCR products. First, it is readily standardized because it is a simple enzymatic process that does not depend on the use of living cells. Second, only a single sequence needs to be determined for each sample. The ease with which clear and reliable sequences can be obtained by direct sequencing depends on the ability of the PCR primers to amplify only the target sequence, and the method used to obtain a template suitable for sequencing.

Association of insulin-dependent diabetes mellitus in Taiwan with HLA class II DQB1 and DRB1 alleles

The allelic constitution at HLA class II DRB1, DQB1, DQA1, and DPB1 loci of IDDM patients from Taiwan was compared with that of ethnically matched nondiabetic individuals by PCR-based DNA typing. Of the three haplotypes found to be positively associated with IDDM in Taiwan, two (DRB1*0301-DQA1*0501-DQB1*0201 and DR4-DQA1*0301-DQB1*0302) appear to be identical to the susceptible haplotypes in Caucasian and black populations, whereas the third haplotype (DR4-DQA1*0301-DQB1*04) has been reported to be positively associated with IDDM only in the Japanese population. The three haplotypes, DRB1*1502-DQA1*0102-DQB1*0601 and DRB1*1201 (or 1202)-DQA1*0501-DQB1*0301 and DRB1*0803-DQA1*0103-DQB1*0601, were negatively associated with IDDM in Taiwan; a protective effect of the last haplotype has not been reported previously. Neither DQ beta non-Asp-57 nor DQA1*0301 alone appears sufficient to account for the HLA-associated susceptibility to IDDM in Taiwan. Also, the DQ alpha beta heterodimer encoded by the alleles DQA1*0301/DQB1*0201, DQA1*0301/DQB1*0302, or DQA1*0501/DQB1*0201 does not explain the susceptibility of a larger fraction of the IDDM patients than the residue at position 57 of the DQ beta chain or DQA1*0301. Finally, the DRB1 alleles appear to affect IDDM susceptibility, although for most haplotypes the effect of individual loci cannot be assessed due to the linkage disequilibrium between the DQ and the DR region.

Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing

Analysis of mtDNA variation using Sanger sequencing does not allow accurate quantification of the components of mtDNA mixtures. An alternative method to determine the specific mixture ratios in samples displaying heteroplasmy, consisting of DNA contributions from several individuals, or containing contamination would therefore be valuable. A novel quantification system for mtDNA mixture analysis has been developed based on pyrosequencing technology, in which the linear relationship between incorporated nucleotides and released light allows quantification of the components of a sample. Within five polymerase chain reaction fragments, seven variable positions in the mtDNA control and coding region were evaluated using this quantification analysis. For all single nucleotide polymorphisms quantified in this study, a linear relationship was observed between the measured and expected mixture ratios. This mtDNA quantification assay is an easy to use, fast and accurate quantification system, with the ability to resolve and interpret major and minor mtDNA components in forensic mixture samples.

Forensic Casework Analysis Using the HVI/HVII mtDNA Linear Array Assay

The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.

A comprehensive polymerase chain reaction-oligonucleotide typing system for the HLA class I A locus

A comprehensive system for genetic typing of the HLA class I A locus is described, based on PCR amplification and typing with nonradioactively labeled SSO probes. Exons 1-3 of the A locus are amplified and typing is performed with a set of 30 nonradioactively labeled oligonucleotide probes. This system resolves 34 of 39 known alleles and 561 (94%) of 595 possible genotypes. Among a sample of 354 individuals from Sweden and China, 97.5% of the genotypes were resolved. Probes were directed preferentially at replacement substitutions in foreign antigen-binding sites, in order to detect not only the known alleles but also new combinations of polymorphic motifs, indicative of previously unrecognized alleles. Three individuals were found with a new combination of polymorphic motifs, suggesting the presence of at least one previously undescribed allele in the populations sampled. This typing system is useful for disease association studies, tissue typing, and in forensic medicine.

Genetic identification of putative remains of the famous astronomer Nicolaus Copernicus

We report the results of mitochondrial and nuclear DNA analyses of skeletal remains exhumed in 2005 at Frombork Cathedral in Poland, that are thought to be those of Nicolaus Copernicus (1473–1543). The analyzed bone remains were found close to the altar Nicolaus Copernicus was responsible for during his tenure as priest. The mitochondrial DNA (mtDNA) profiles from 3 upper molars and the femurs were identical, suggesting that the remains originate from the same individual. Identical mtDNA profiles were also determined in 2 hairs discovered in a calendar now exhibited at Museum Gustavianum in Uppsala, Sweden. This calendar was the property of Nicolaus Copernicus for much of his life. These findings, together with anthropological data, support the identification of the human remains found in Frombork Cathedral as those of Nicolaus Copernicus. Up-to-now the particular mtDNA haplotype has been observed only 3 times in Germany and once in Denmark. Moreover, Y-chromosomal and autosomal short tandem repeat markers were analyzed in one of the tooth samples, that was much better preserved than other parts of the skeleton. Molecular sex determination revealed that the skeleton is from a male individual, and this result is consistent with morphological investigations. The minimal Y-chromosomal haplotype determined in the putative remains of Nicolaus Copernicus has been observed previously in many countries, including Austria, Germany, Poland, and the Czech Republic. Finally, an analysis of the SNP located in the HERC2 gene revealed the C/C genotype that is predominant in blue-eyed humans, suggesting that Copernicus may have had a light iris color.

Forensic analysis of autosomal STR markers using Pyrosequencing.

Short tandem repeats (STRs) are highly variable, and therefore routinely used in forensic investigations for a DNA-based individual identification. The routine assay is commonly performed by size separation using capillary electrophoresis, but alternative technologies can also be used. In this study, a Pyrosequencing assay was developed for analysis of STR markers useful in forensic DNA analysis. The assay was evaluated for 10 different STR loci (CSF1PO, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539 and Penta E) and a total of 114 Swedish individuals were genotyped. This genotyping strategy reveal the actual sequence and variant alleles were seen at several loci, providing additional information compared to fragment size analysis. At the D13S317 locus a T/A SNP located in the last repeat unit was observed in 92% of the genotypes. Moreover, an upstream flanking SNP at locus D7S820, a SNP within the repeats at D3S1358 and D8S1179 and a deletion in the flanking region at locus D5S818 were observed. The Pyrosequencing method was first developed for SNP typing and sequencing of shorter DNA fragments but the method also provides an alternative method for STR analysis of less complex repeats. This assay is suitable for investigation of new markers, a rapid compilation of population data and for confirmation of variant and new alleles.