Multiplexed short tandem repeat polymorphisms of the Weber 8A set of markers using tailed primers and infrared fluorescence detection

Abstract

Short tandem repeat polymorphism (STRP) markers have become important reagents for mapping genetic diseases. These markers are available as screening sets, which are located in all chromosomes at discrete intervals, allowing the entire genome to be analyzed. Mapping studies that include many individuals in the analysis necessitate the production of large numbers of genotypes. In an effort to increase the efficiency and lower the cost of using these STRP screening sets, we have divided the amplification primers of the Weber 8A screening set into groups that can be amplified in single polymerase chain reaction (PCR) amplification reactions, resulting in a reduction of both time and cost. Fluorescently-labeled amplification products were produced using a three primer reaction. The forward STRP amplification primer for each marker contained a 19 bp sequence at the 5' end. A fluorescently-labeled primer, with a sequence identical to the 19 bp tail, was added to the amplification reaction as the sole source of fluorescent label. The STRP banding pattern is detected using an automated fluorescent DNA sequencer. Use of this multiplexed genomic screening set should greatly enhance the mapping of human disease loci.

Microsatellite polymorphism in the sexually transmitted human pathogen Trichomonas vaginalis indicates a genetically diverse parasite

“…First, we adapted a ‘tagged primer’ technique [52, 53] by adding a 19 nt tag to the 5′ end of each forward primer, designed to be recognized by a fluorescently tagged universal primer. Primers were added in a 1:40:40 ratio (locus specific forward primer: locus specific reverse primer: tagged universal primer) to perform semi-nested PCRs in which priming was done first with locus-specific reactions, which were then used as a template for reactions with the labeled primer.…”

“…During our MS marker development, we employed a cost-saving method that utilized one universal tagged primer to label PCR amplicons of the MS loci [52, 53, 55], making the technique accessible to more researchers, particularly those in resource-limited settings. The availability of these reproducible markers will allow comparison and merging of data sets from different research projects, enabling a more comprehensive investigation of the population diversity of T. vaginalis through better representation of widespread geographical locations, population demographics, and overall parasite diversity.…”

An Integrated Genetic and Physical Map for the Malaria Vector Anopheles funestus

“…Initially we used published primer sets (Sinkins et al 2000;Cohuet et al 2002;Sharakhov et al 2002;Soremekun et al 2004) to individually amplify 72 microsatellite markers from the parental and F 1 A. funestus genomic DNA. A tailed primer system, described by Oetting et al (1998), was used to label the PCR products with a fluorescent dye to reduce the genotyping costs. PCR products were first run on 1.5% agarose gels and the products of successful PCR reactions were sized using the Beckman CEQ8000 fragment analysis software.…”

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“…For the apparatus to detect PCR products, one primer in every pair of microsatellite primers must be fluorescently labelled. To reduce the cost associated with synthesis of fluorescently labelled primers, we used the "tailed primer" method [34,35]; that is, the forward primer for each microsatellite locus was synthesized with an additional 19 bp sequence (5' CACGACGTTGTAAAACGAC 3') added to the 5' end of the primer. A third primer with the same 19 bp sequence was directly labelled with the fluorescence and was used as the sole type of labelled primer for the detection of all microsatellite alleles.…”