Donald J. Kessler, Sandie Yu, and Dean Burgi
Genomyx Corporation, 353 Hatch Drive, Foster City, CA 94404

× Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø

Short tandem repeat (STR) loci consist of tandemly repeated 3-7 base pair sequences. The variation in the number of copies of this repeated sequence motif provides a valuable source of genetic markers. Their abundance, one trinucleotide or tetranucleotide repeat locus every 15 kb in the human genome¹, and their ability to be amplified using PCR and flanking sequence primers, makes them ideal for applications in forensic analysis, paternity determination, human cell line identification and the monitoring of bone marrow transplants. Most STR systems yield a defined PCR amplification product which can be analyzed following electrophoresis by either autoradiography (isotopic), staining (silver) or fluorescence.

Fluorescence provides a number of advantages over the other labeling methods, these are: 1) Fluorescent compounds and substrates have a longer shelf life than radioisotopes (which must be used immediately to avoid radiolytic decay); 2) safety, handling and disposal issues are significantly less demanding and expensive than those for radioisotopes and silver staining; 3) fluorescent detection methods are much faster than autoradiography and silver stain development; 4) the fluorescent signal is linear over a wider concentration range, allowing more flexibility in quantitative analysis; 5) multiple fluorescent color selection allows for greater throughput where multiple variables must be measured simultaneously; 6) background can be minimized by specific selection of the fluorescent label; and 7) the use of electronic data collection, including the use of spectral software for fragment size determination and calling of allele and genotype.

In the past, the most commonly used labeling techniques for STR analysis have been isotopic and silver staining. Today, the use of fluorescence offers a safer alternative to labeling and detection. The use of genetic markers labeled with fluorophores (fluorescent coumpounds) for STR assays has increased tremendously over the past couple of years as nucleic acid chemists have successfully designed fluorophore modified nucleic acid substrates.

These modified substrates can be incorporated either through solid phase synthesis (i.e., fluorescent phosphoramidites for the synthesis of modified oligonucleotide primers which can be used in PCR [polymerase chain reaction, Hoffman-LaRoche, Inc., Nutley, N.J.] amplification reactions), or through enzymatic reactions which incorporate fluorophore modified dNTPs (e.g., FL-dUTP² [deoxy-uridine triphosphate] incorporation by Taq polymerase during PCR). Internal incorporation of a fluorophore modified dNTP offers the researcher the luxury of utilizing less expensive unmodified primers, thus alleviating the need to resynthesize or purchase fluorophore modified primers.

In this work, a method to internally label PCR products with multiple colored fluorescent dNTP (deoxy-nucleotide triphosphate) compounds is described and demonstrated for STR analysis. PCR-amplified fluorescent STR fragments, which were internally labeled by adding fluorescent dCTPs ([R110]dCTP, [R6G]dCTP, [TAMARA]dCTP; Perkin-Elmer/Applied Biosystems, Inc., Foster City, CA and [Cy5]dCTP; Amersham Corp., Arlington Heights, IL) to the PCR mixture were applied on a denaturing polyacrylamide gel and electrophoresed on the GenomyxLR (Genomyx Corp., Foster City, CA) sequencer which has a temperature controlling system. The gel was imaged using the GenomyxSC (Genomyx Corp., Foster City, CA) fluorescent scanner.