Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction - amplified short tandem repeats during denaturing capillary electrophoresis

Hahn, Meinhard; Wilhelm, Jochen; Pingoud, Alfred

doi:10.1002/1522-2683(200108)22:13<2691::aid-elps2691>3.0.co;2-s

Cited by 44 publications

(39 citation statements)

References 24 publications

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“…Such inconsistencies are mainly due to the large variety of automatic sequencing machines used, each providing different gel migration, fluorescent dyes and allele calling software possibilities. For instance, the type of fluorescent dye used will influence migration, and moreover, this will depend on the length and sequence of the DNA strand [30]. In some cases, even the use of multiple standard samples loaded on gels does not solve problems, particularly when large size differences between alleles exist.…”

Section: Technical Considerationsmentioning

confidence: 99%

A review on SNP and other types of molecular markers and their use in animal genetics

et al. 2002

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-During the last ten years, the use of molecular markers, revealing polymorphism at the DNA level, has been playing an increasing part in animal genetics studies. Amongst others, the microsatellite DNA marker has been the most widely used, due to its easy use by simple PCR, followed by a denaturing gel electrophoresis for allele size determination, and to the high degree of information provided by its large number of alleles per locus. Despite this, a new marker type, named SNP, for Single Nucleotide Polymorphism, is now on the scene and has gained high popularity, even though it is only a bi-allelic type of marker. In this review, we will discuss the reasons for this apparent step backwards, and the pertinence of the use of SNPs in animal genetics, in comparison with other marker types.SNP / microsatellite / molecular marker / genome / polymorphism

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Section: Technical Considerationsmentioning

confidence: 99%

A review on SNP and other types of molecular markers and their use in animal genetics

et al. 2002

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“…Strain IHEM 9670 was run as a control in each experiment. As allele sizing depends on dyes and the analyzer used for electrophoresis, results were expressed as the exact size of the sequence (determined by sequencing of each representative allele for each locus) to allow further interlaboratory comparisons of MLVA results (13,20). The reproducibility and stability of the method were assessed as described elsewhere (25 .…”

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confidence: 99%

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

et al. 2007

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A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power (D ‫؍‬ 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.Candida glabrata has recently emerged as a major pathogen, causing mucosal and systemic infections (10,17). Several methods, such as electrophoretic karyotyping, restriction enzyme analysis, Southern blotting with probes, randomly amplified polymorphic DNA, and multilocus sequence typing (MLST), have been used to distinguish and type C. glabrata isolates (2,7,8,14,21,28). Microsatellite polymorphism analysis has been widely used for typing fungi (3,4,11,15,18,25), and this could be an alternative, easy-to-perform, reproducible method suitable for large-scale studies of C. glabrata epidemiology. Recently, Foulet et al. described three polymorphic microsatellite markers to investigate the delineation of clinical C. glabrata isolates (12). The discriminatory power of this method was good but not optimal. The aim of our study was to assess a microsatellite-based multiple-locus variable-number tandemrepeat analysis (MLVA) using new markers for C. glabrata typing.One hundred twenty-seven C. glabrata strains were analyzed, including four reference strains, 98 independent clinical isolates, and 25 epidemiologically related isolates. These 25 were from the blood cultures and peripheral site isolates of eight patients with C. glabrata candidemia. Genomic DNA was extracted by boiling with Chelex resin as previously described (6, 23). Six microsatellite markers were selected from the C. glabrata DNA sequences available in the GenBank database (29). Primer sequences were designed with Primer3 software (24), and locations in the C. glabrata genome were determined with the Genolevures database (http://cbi.labri.fr/Genolevures/) ( Table 1). For each primer set, PCRs were performed with a 20-l final volume containing 1 l of DNA, each deoxynucleoside triphosphate at 200 M, a forward primer and a 5Ј-dyelabeled reverse primer at 0.25 M each, and 1 U of Taq DNA polymerase (Promega, Madison, WI). The amplification conditions were 5 min at 95°C, followed by 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 45 s and then a final step of 72°C for 10 min. For any given isolate, amplicons of each PCR were pooled before multiplex fragment sizing with a Ceq 8000 Genetic Analyzer (Beckman Coulter, Fullerton, CA). Strain IHEM 9670 was run as a control in each experiment. As allele sizing depends on dyes and the analyzer used for electrophoresis, results were expressed as the exact size of the sequence (determined by sequencing of each representative allele for each locus) to allow further interlaboratory comparisons of MLVA results (13,20). The reproducibility and stability of the method were assessed as described elsewhere (25) . Primer specificity was also tested against the sequ...

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“…Conditions employed in Germany: Two combinations of primers were used after Hahn et al (1993Hahn et al ( , 2001. PCR conditions were according to Hahn et al (2001).…”

Section: Pcr Conditionsmentioning

confidence: 95%

“…Conditions employed in Germany. Native and denaturing PAGE, and, denaturing capillary electrophoreses (Hahn et al 2001).…”

Section: Separation Of Pcr Productsmentioning

confidence: 99%

The STR polymorphism (AAAAT)n within the intron 1 of the tumor protein 53 (TP53) locus in 17 populations of different ethnic groups of Africa, America, Asia and Europe

Azofeifa

Hahn

Ruiz

et al. 2014

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Abstract:The STR (AAAAT) n within intron 1 of the TP53 locus was screened in 17 populations from 3 main ethnic groups: Europeans, Asiatics, and Africans, and from the hybrid population of Costa Rica (1968 samples). Three alleles, 126/7 (bp/copies of the repeat), 131/8 and 136/9 were the most prevalent in all populations. Other alleles rarely reached frequencies of 10% or higher. Observed heterozygosities ranged between 0.351 and 0.829. Patterns of diversity fit well with both the geographic origin of the samples and the history of the populations screened. A statistical test suggests that single-step mutational events have been the main mechanism producing new alleles at this locus. Fixation indexes (R ST ) for this marker showed an effect of population subdivision on divergence only within the Asiatic group; they were insensitive at the level of major ethnic groups as well as within Africans and within Europeans. Rev. Biol. Trop. 52(3): 645-657. Epub 2004 Dic 15.

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Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction - amplified short tandem repeats during denaturing capillary electrophoresis

Cited by 44 publications

References 24 publications

A review on SNP and other types of molecular markers and their use in animal genetics

A review on SNP and other types of molecular markers and their use in animal genetics

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

The STR polymorphism (AAAAT)n within the intron 1 of the tumor protein 53 (TP53) locus in 17 populations of different ethnic groups of Africa, America, Asia and Europe

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