James M. Robertson, Janet Ziegle and Michael Wentz
Perkin-Elmer Corporation, Applied Biosystems Division, Foster City, CA
Recently, a capillary electrophoresis based-instrument was developed for the specific analysis of DNA (ABI PRISM™ 310 Genetic Analyzer). The advantages of the instrument include a rapid separation of DNA fragments of around 300 nucleotides in length with a resolution of 2 base pairs. This specification means that the alleles of most STR loci considered useful for forensic DNA analysis can be unambiguously identified in about 20 minutes without the necessity of pouring and loading gels. Following the PCR, the samples can be mixed with denaturant, dissociated, and placed in the automatic sampler device and analyzed with a polymer and buffer containing urea, as in the case of STR analysis. In addition, when it is desired to analyze large amplimers such as those from VNTRs, the samples are simply diluted with water and placed in the loader without denaturant, and a native polymer mixture is utilized for the separation of the DNA fragments.
Here, we present preliminary studies on the separation of the alleles of the STR loci vWA, THO1, F13A1, FES/FPS, and SE33. The 3.2 and 4 alleles of the F13A1 allelic ladder, which differ by 2 nucleotides in length, are well resolved in about 15 minutes. Since the instrument is capable of distinguishing at least four colors, alleles that are overlapping in size can be detected, because we utilize a dye that fluoresces in blue for one locus and a dye that fluoresces in green for the other locus. Examples will be shown for the loci vWA and TH01, which have alleles that overlap in the region 158-167 bp, and of the loci F13A1 and FES/FPS, which have alleles of similar size in the region 206-234 bp. The polymer concentration in the capillary can be easily changed for good resolution of SE33 alleles, which have a relatively large size (up to 320 base pairs in about 22 minutes).
By utilizing a capillary containing a polymer without denaturant, one can obtain separations of large DNA fragments similar to that observed with agarose gels. Examples will be shown for the VNTR D1S80, with alleles that approach 800 base pairs in length. The sizes obtained with the associated data analysis software vary by only a few base pairs from those obtained with agarose analysis. The only difference is that the separation is achieved in less than 20 minutes when using the capillary electrophoresis instrument.
The features of the capillary electrophoresis instrument include variable injection times. This allows the analyst to detect weak samples with high sensitivity by employing a longer injection time than used for strong samples.
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