Primer fluorescently labeled

The STR data generated at NIST were based on amplifying i.o ng of the genomic DNA with fluorescent labelled primers. The PCR amplified products were analyzed by slab gel electrophoresis followed by imaging with a Molecular Dynamics Fluorl-mager 595 or by capUlary electrophoresis using a PE-ABI 310 Genetic Analyzer. 

Microsatellites are hypervariable co-dominant loci composed of arrays of 2-9 bp repeating motifs. Differences in the number of repeat motifs in an array define microsatellite polymorphisms. Method development requires the identification of microsatellite loci, and for each locus, the design of PCR primers to anneal to conserved regions flanking the microsatellite. Analysis involves PCR amplification with fluorescently labeled primers followed by electrophoresis to distinguish microsatellite alleles of different array size. 

One advantage of fluorescently labeled primers over dsDNA dyes is that multiplexing is possible. However, with both dsDNA dyes and labeled primers, reaction specificity depends on the specificity of the primers. Any double-stranded product that is formed will be detected, including primer-dimers. Therefore, hot start techniques, temperature discrimination by collecting real-time data at a high temperature, and melting curve analysis to confirm the desired product are useful. 

Kirov, G., Stephens, M., Williams, N., O Donovan, M., and Owen, M. (2000) Automated genotyping of single-nucleotide polymorphisms by extension of fluorescently labelled primers analysis of individual and pooled DNA samples. 

Fig. 7 Principle of the T-RFLP technique. Following extraction of nucleic acids, PGR amplification is performed using one fluorescently labeled primer. The amplified products are then subjected to restriction digestion, which leads to the generation of fluorescently labeled fragments of different lengths. The fragments are separated on a sequencing gel...

Figure 2 A schematic view of multiplex PCR analysis of STRs. In a test tube, some STRs from the sample s DNA (A) are amplified by PCR (B) using fluorescent-labeled primers. After the addition of an internal standard (red-labeled fragments) (C) the DNA fragments obtained at the end of the PCR are separated by capillary electrophoresis according to their size (D) and detected at the end of the capillary. Each peak (E) is then electronically labeled with the name of the corresponding allele. The profiles in blue, green, and black present the alleles detected for the STRs VWA, D21S11, andTHOI, respectively. The red profile displays two peaks from the internal standard.

The most successful applications of CE-LIF detection have been in the analysis of carbohydrates and nucleic acids. Carbohydrates can be derivatized with agents such as aminopyrene trisulfonic acid, which introduces a fiuorophore and also confers electrophoretic mobility to the analyte. Double-stranded nucleic acids can be detected using intercalating dyes and by covalent attachment of nucleotides carrying fluorophores. The fluorescently labeled primers or chain terminating nucleotides which are widely used in automated slab gel-based DNA sequencers are also used in capillary-based sequencers. In one multicapillary sequencer, a novel sheath flow system is used for LIF excitation of separated bands with imaging of emitted fluorescence on a charge-coupled device (CCD) detector. 

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