An Improved Method for Post-PCR Purification for mtDNA Sequence Analysis

Dugan, Kerri A.; Lawrence, Helen S.; Hares, Douglas R.; Fisher, Cindy L.; Budowle, Bruce

doi:10.1520/jfs15459j

Cited by 65 publications

(50 citation statements)

References 6 publications

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“…Negative controls, which contained water instead of template DNA, were run in parallel in each run. The PCR product was purified by an enzymatic method, modified from the method of Dugan et al (11), with exonuclease I (New England Biolabs GmbH, Frankfurt-Hoechst, Germany) and shrimp alkaline phosphatase (Amersham Pharmacia Biotech, Freiburg, Germany). Briefly, 5 l of the PCR product was incubated with 1 U of each enzyme at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

et al. 2008

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Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.

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Section: Methodsmentioning

confidence: 99%

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

et al. 2008

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“…A 345 bp portion of the mitochondrial DNA control region was amplified using the primers t-PRO and Primer-2 from Yoshida et al (2001), using the same PCR conditions as for the microsatellite loci, and an annealing temperature of 55 1C. After amplification, primers and unincorporated dNTPs were degraded using EXOSAP-IT (Dugan et al, 2002), and products were sequenced using the DYEnamic dye terminator kit (GE Healthcare, Piscataway, NJ, USA). Products were size-separated and visualized on a MegaBACE 1000 (GE Healthcare).…”

Section: Molecular Methodsmentioning

confidence: 99%

Detecting recent speciation events: the case of the finless porpoise (genus Neophocaena)

Wang

Frasier

Yang³

et al. 2008

Heredity

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Recent speciation events provide important insights into the understanding and conservation of Earth's biodiversity, representing recent adaptations to a changing environment and an important source of future evolutionary potential. However, the most frequently applied criterion for molecularbased speciation investigations, that of reciprocal monophyly of mitochondrial sequences, overlooks recent speciation events where insufficient time has passed for fixed molecular differences to develop between putative species. Two morphologically distinguishable forms of finless porpoise (genus Neophocaena) exist in sympatry in the strait of Taiwan, however the taxonomic relationship of these different forms is controversial. To test the hypothesis that the two forms represent different species, a study was conducted based on morphological characters and microsatellite and mitochondrial markers. The data suggest that the two forms are highly differentiated in terms of both morphology and genetic characteristics, despite being sympatric, and therefore represent different species as defined by the biological species concept. Moreover, the two forms appear to have been reproductively isolated since sharing a common ancestor prior to the last major glaciation event B18 000 years ago. However, this represents an insufficient amount of time for reciprocal monophyly to have developed, and thus previous studies based on this criterion have overlooked this speciation event and resulted in incorrect taxonomic classification of these forms.

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“…Mitochondrial COI region sequencing: Amplified PCR products were subsequently cleaned by the exo-SAP method (Dugan et al 2002). Five µl of PCR product, 0.7 µl of exonuclease I, 10x buffer (New England Biolabs, MA, USA), 0.5 µl of exonuclease I and 5.3 µl of nanopure water were incubated at 37°C for 30 min before being denatured at 80°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Molecular identification of two gobi fishes of Bangladesh using cytochrome oxidase subunit I (CoI) gene sequences

Tabassum

Jahan

Latifa

2017

Bangladesh J. Zool.

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DNA barcoding has been proposed as a means of quick species identification using a short standardized segment of DNA. Two species (Eleotris fusca and Glossogobius giuris) from the family Gobiidae and Eleotridae were selected for DNA barcoding using samples collected from different regions of Bangladesh. Cytochrome Oxidase Subunit I (COI) gene was sequenced from two different gobi fishes and compared with two previously published similar sequences from the genera Eleotris and Glossogobius. Multiple sequence alignment and the molecular systematic study were performed. The DNA barcode technique identified the two species. The study provides a good example of how DNA barcoding can build upon its primary mission of species identification and use available data to integrate genetic variation investigated at the local scale into a global framework.

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An Improved Method for Post-PCR Purification for mtDNA Sequence Analysis

Cited by 65 publications

References 6 publications

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

Detecting recent speciation events: the case of the finless porpoise (genus Neophocaena)

Molecular identification of two gobi fishes of Bangladesh using cytochrome oxidase subunit I (CoI) gene sequences

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