3100 Genetic Analyzer From Applied Biosystems

Short Tandem Repeats (STRs) are accepted markers for human forensic analysis and DNA fingerprinting. In bacterial systems, Variable Numbers of Tandem Repeats (VNTRs) and Amplified Fragment Length Polymorphisms (AFLPs) are also used extensively in molecular epidemiology and phylogenetic studies. Polyacrylamide gels had been the gold-standard for these types of analyses. However, efforts to create human genetic databases have revealed limitations in slab-gel systems that necessitate the use of instrumentation more amenable to automated and high-throughput analyses.

The technology employed is fairly straightforward. Most capillary array instruments separate DNA by molecular weight through the application of a current across a non cross-linked polymer matrix. This allows simultaneous resolution of 1 to 96 DNA samples within separate capillaries, depending on the instrumentation used. ABIs 3100 capillary electrophoresis instrument uses a 16-capillary array. This technology also requires the use of a conjugated fluor (e.g., 6-FAM, TET, or HEX) with varying absorbance and emission spectra for amplicon detection. Typically, one of the primers in the PCR reaction is labeled with such a fluor which is incorporated in the amplicon generated during PCR.

We’ve used this system in conjunction with several different size standards including an in-house custom size standard with favorable results. As with most capillary systems, the amplicon size derived from capillary electrophoresis will differ slightly from the actual amplicon size derived from sequence analyses. However, run to run variation of the same amplicons is negligible on the ABI3100. We’ve currently standardized on a five-dye format with ABIs 1200 LIZ® and 500 LIZ® size standards for the majority of our fragment analyses. We’ve found that these commercially available size standards are robust and allow good resolution for most applications. Custom size standards can also be used with this system; however, the majority of available dyes (such as ROX™) will limit you to a four-dye system. We’ve also standardized on Genemapper™ software for downstream fragment analysis. GeneMapper™ is geared towards human STRs and microsatellites, but with some amending of the software parameters, it will recognize larger repeat motifs such as bacterial VNTRs. Additionally, we’ve also used this instrument for Single Nucleotide Polymorphims (SNPs) by using ABI’s SNaPshot method, as well as our own in-house multiplex variation.

The 3100s’ 16-capillary array is sufficient for our current throughput. For most analyses, it takes about 6 hours to run an entire 96-well plate (six injections). A 96-well version (3730 DNA Analyzer) is also available for higher throughput labs. It’s not the least expensive platform for capillary electrophoresis, but it certainly is among the best in terms of precision, accuracy, sensitivity and resolution. This is about the most versatile piece of equipment you could hope to have.