Short Tandem Repeat Typing by Capillary Array Electrophoresis: Comparison of Sizing Accuracy and Precision Using Different Buffer Systems

Vainer, Marina; Enad, Shellee; Dolnı́k, Vladislav; Xu, Diane; Bashkin, John; Marsh, Michele; Tu, Oahn; Harris, Dennis W.; Barker, David; Mansfield, Edwin

doi:10.1006/geno.1997.4608

Cited by 31 publications

(19 citation statements)

References 28 publications

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“…Even in these cases, the differences were inferred to be caused by switches from SE machines to CE machines (8) or were hinted at rather than stated directly (27). In contrast, we in this study, and others outside the field of microbiology, have shown that different CE machines and running conditions will affect the sizing of alleles (10,25,33,38,40,41). Another microbiology study made the very general statement that separation techniques have been shown to produce different results for the same locus and therefore that special care must be taken when standardizing typing data (34).…”

Section: Discussionmentioning

confidence: 60%

“…Switching to another fluorescent label can alter the mobility of a DNA molecule by Ϯ0.5 bases (10), and temperature fluctuations in the laboratory housing the machine can alter mobility by up to 0.7 bases, with a temperature difference of 5°C (9). Other, potentially more serious problems include migrational deviations of the internal size standard, and we have identified one study which used an internal size standard that has been recommended not to be employed with CE machines (1,38). The calibration method used is also important, and the local Southern approach is recommended (16).…”

Section: Discussionmentioning

confidence: 99%

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A Cautionary Tale: Lack of Consistency in Allele Sizes between Two Laboratories for a Published Multilocus Microsatellite Typing System

2007

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For species with low genetic diversity, typing using the differences in PCR fragment length resulting from variations in numbers of short tandem repeats has been shown to provide a high level of discrimination. This technique has been called multilocus microsatellite typing (MLMT) or multiple-locus variable-number tandem repeat analysis, and studies usually employ genetic or sequence analyzers to size PCR fragments to a high degree of precision. We set out to validate one such system that has been developed for Aspergillus fumigatus . The sizes of the alleles were compared both by sequencing and from two genotyping laboratories, where they used capillary electrophoresis (CE) for sizing. Size differences of up to 6 bases were found between the actual sizes reported by sequencing and the sizes reported by CE. In addition, because the two genotyping laboratories used different machines and running conditions, differences of up to 3 bases were identified between them. As the microsatellite markers used differ by repeat units of 3 or 4 bases, it was not possible to assign PCR fragments to the correct alleles without confirming the sizes of a range of alleles by direct sequencing. Lines of best fit were plotted for each CE machine against actual sizes and will therefore enable unsequenced PCR fragments to be assigned to the correct alleles. This study highlights the care required to ensure that an MLMT system undergoes a suitable correction procedure before data can be merged between different laboratories involved in the typing of individual species.

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

A Cautionary Tale: Lack of Consistency in Allele Sizes between Two Laboratories for a Published Multilocus Microsatellite Typing System

2007

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“…An improved system carried 96 capillaries, where the introduction of samples was done by electrokinetic injection. At an average field strength of 185 V/cm in polyacrylamide capillaries, of a 350 bp standard was separated in only 25±30 min and excellent correlation between computed and actual fragment sizes was observed [15].…”

Section: Alternative Methods Of Pcr Product Separationmentioning

confidence: 96%

Molecular genetic diagnosis of de novo and recurrent bladder cancer

1999

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“…[21][22][23] A new analytical technique, CE has been introduced into the field. 24 CE is an attractive alternative to slab gel electrophoresis in DNA analysis for the following reasons: faster separation and higher resolution, the possibility of automating sample loading and semiquantitative analysis of results. In addition to this, CE eliminates the laborintensive, time-consuming procedure of electrophoretic DNA preparation by slab gel.…”

Section: ·3 Capillary Electrophoresis For Dna Sequencingmentioning

confidence: 99%

The Human Genome Project: the Role of Analytical Chemists

Thangadurai¹

2004

ANAL. SCI.

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Short Tandem Repeat Typing by Capillary Array Electrophoresis: Comparison of Sizing Accuracy and Precision Using Different Buffer Systems

Cited by 31 publications

References 28 publications

A Cautionary Tale: Lack of Consistency in Allele Sizes between Two Laboratories for a Published Multilocus Microsatellite Typing System

A Cautionary Tale: Lack of Consistency in Allele Sizes between Two Laboratories for a Published Multilocus Microsatellite Typing System

Molecular genetic diagnosis of de novo and recurrent bladder cancer

The Human Genome Project: the Role of Analytical Chemists

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