ReseqChip: Automated integration of multiple local context probe data from the MitoChip array in mitochondrial DNA sequence assembly

Thieme,M.; Lottaz,C.; Niederstätter,H.; Parson,W.; Spang,R; Oefner,P.J.; Background The Affymetrix MitoChip v2.0 is an oligonucleotide tiling array for the resequencing of the human mitochondrial (mt) genome. For each of 16,569 nucleotide positions of the mt genome it holds two sets of four 25-mer probes each that match the heavy and the light strand of a reference mt genome and vary only at their central position to interrogate all four possible alleles. In addition, the MitoChip v2. [Read More]

Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences

Fendt,L.; Zimmermann,B.; Daniaux,M.; Parson,W.; ABSTRACT: BACKGROUND: It has been demonstrated that a reliable and fail-safe sequencing strategy is mandatory for high-quality analysis of mitochondrial (mt) DNA, as the sequencing and base calling process is prone to error. Here, we present a high quality, reliable and easy handling manual procedure for the sequencing of full mt genomes that is appropriate for laboratories where fully automated processes are not available. RESULTS: We amplified whole mitochondrial genomes as two overlapping PCR-fragments comprising each about 8500 bases in length. [Read More]

Standards zu forensisch-molekulargenetischen Untersuchungen, Massenunfälle und Katastrophen

Lessig,R.; Aspinall,L.; Krumm,P.; Wiegand,P.; Hohoff,C.; Steinlechner,M.; Roewer,L.; Edelmann,J.; Bastisch,I.; Die Identifizierung von Opfern eines Massenunfalls oder einer Katastrophe stellt nach wie vor für alle Beteiligten eine große Herausforderung dar. Eine der primären Identifizierungsmethoden ist die DNA-Analyse. Damit bildet sie einen wesentlichen Beitrag im Rahmen dieser Untersuchungstätigkeit. Die Erfahrungen aus dem Tsunami 2004 und den Unglücken der letzten Monate haben gezeigt, dass es nach wie vor Handlungsbedarf auf diesem Gebiet gibt. Je nach Opferlage und Anzahl der Opfer gehört es dazu, dass die molekulargenetischen Untersuchungen in einem mehr oder weniger großen Umfang durchgeführt werden. [Read More]

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples

Eichmann,C.; Parson,W.; The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. [Read More]