Katherine Lazaruk, P. Sean Walsh, James Robertson and Jeanette Wallin
Perkin-Elmer Applied Biosystems, Foster City, CA

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Two new multiplex STR amplification and typing kits are currently under development at PE Applied Biosystems for laboratories with high throughput needs. Each kit coamplifies 9 STR loci and amelogenin (some loci shared between the two kits) in a single PCR reaction using the new AmpliTaq Gold DNA Polymerase. All 10 PCR amplification products can subsequently be detected in a single lane of an ABI PRISM™ 377 DNA Sequencing gel or in a single injection on an ABI PRISM 310 capillary electrophoresis instrument. The power of discrimination of each 9-plex is 1 in 3 billion and 1 in 72 billion, respectively. Each kit employs AmpliTaq Gold as the thermostable polymerase and therefore uses an invisible hot start in each PCR reaction. AmpliTaq Gold allows for very efficient amplification of a larger number of loci in a single reaction tube while virtually eliminating the formation of primer-dimer and/or non-specific amplification products.

Each 9-plex is optimized for the standard ABI PRISM multi-color detection using a CCD camera. The CCD camera technology allows for future incorporation of new fluorescent dyes into the existing dye sets, thus expanding the potential number of loci that can be detected in a single lane. There is a very active program at ABI for the development of new dyes to fill the "holes’ in the detection spectrum of the CCD camera. Our first two 9-plexes have incorporated a new fluorescein dye as a replacement for Tamra. The new dye has both a higher excitation efficiency and a blue-shifted emission spectrum as compared to Tamra, thus the detection sensitivity of the multiplex has been increased. With the development of other new fluorescent dyes, it will be possible to coamplify even greater numbers of loci in a single PCR reaction for detection in a single gel lane.

ABI is committed to providing ever increasing throughput capabilities. The first expansion is through the 373 and 377 XL upgrades, where the instruments will be capable of running up to 64 lanes per gel. The gel tracking software is being modified to allow for these extra lanes and for better quantitation of the DNA in a single band on the gel. Later throughput expansions may take the form of a microchanneling adaptation of the existing 377 platform that merges the capillary and gel electrophoresis technologies into one hands-off automated system.

All of the STR data generated by the DNA sequencers and the 310 capillary electrophoresis instrument is automatically analyzed and genotyped using the latest Genotyper software. The new version of Genotyper software uses the allelic ladder sizes on each gel to determine genotypes. Genotyper essentially "manages" the results (i.e. the more than 300 loci per gel) and maintains the data in computerized form for easy downloading into CODIS. The genotypes can be displayed in a variety of tabular forms for easy interpretation.

Results using all of these tools for high throughput analysis will be discussed during the presentation.