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ANALYTICAL CURRENTS
Quicker than thieves Forensic databases containing stretches of repetitive DNA sequences called short tandem repeats (STRs) are cropping up all over the world for use in human identification. Traditional slab-gel and capillary electrophoretic methods for the analysis of STRs, however, are too slow to keep pace with criminals, creating a backlog of samples. Daniel Ehrlich and co-workers at the Whitehead Institute for Biomedical Research have developed a microfabricated electrophoretic device that might be just what is needed to increase the throughput of DNA analysis. Although the device contains only a single channel, it can simultaneously analyze eight STR loci in 2 min. In comparison, capillary and slab-gel techniques take about 1-3 h per run, even with two-color multiplexing. The speed of the new system is attributed to its short separation distance. The detector, in this case a dual-wavelength laserinduced fluorescence detector, can be placed anywhere along the 11.5-cm separation channel. Decreasing the separation distance decreases the analysis
Validating kinetic RT-PCR The reverse transcriptase-polymerase chain reaction (RT-PCR) is used to amplify cDNA fragments made from mRNA In "real-time" or "kinetic" RT-PCR methods, which determine the expression levels of genes, the key is determining how much product accumulates and how quickly. Now, P. Mickey Williams and colleagues at Genentech compare two approaches to calculating the fold increase (e.g., 10-fold versus 20-fold) and describe the validation of a kinetic RT-PCR technique. The RT-PCR technique chosen by the researchers uses a probe labeled with a fluorescent dye and a quencher. When the probe is intact, the quencher and dye are close to each other, keeping the fluorescence level very low. When the probe hy-
time. Speed, however, comes at the expense of resolution. Generally, 2 cm is the minimum distance required for separating DNA fragments (100-400 bases) with fourbase resolution. In some cases, however, such when a microvariant allele is present, singlebase resolution is Electrophoretic microfabricated device with a dual-wavelength laser-induced fluorescence detector. (Adapted with permission. necessary to Copyright 1999 Academic Press.) achieve unambigidentification Using only 2 cm of the The researchers are currently deseparation channel the authors observed veloping an eight-channel microfabripeak broadening in a sample mixed with a cated device, which is expected to hanstandard that contained allele 10 but not aldle 384 multiplexed STR samples per lele 9 3 suggesting the presence of the miday. Their ultimate goal is to have a crovariant allele 9 3 By using the entire 96-channel device, capable of handling 11 5-cm length of the channel thev were 4068 samples per day, which could able to discriminate between the 9 3 and meet the demands of forensic labora10 alleles and the analysis time onlv intories. (Anal. Biochem. 1999, 270, creased to 10 min 148-52) bridizes to its target, however, the polymerase that carries out the PCR reaction cleaves the probe, separating the quencher from the dye and generating a stronger fluorescence signal. The total fluorescence signal is cumulative, growing as each probe is cleaved. The number of reaction cycles needed for the signal to become detectable (C() is determined, and from that, the initial amount of mRNA is calculated. Williams and co-workers looked at the expression of two genes, c-fos and atrial natriuretic factor (Anfi, ,n a aat cell line eesigned to model cardiac hypertrophy. After validation of the sample preparation and verification of glyceraldehyde-3-phosphate dehydro(Gapdh) )s the "housekeeping" gene use(j as a normalization factor the researchers measured the expression levels for a series of dose titrations for each of four hypertrophy agents
The fold increase was calculated using the traditional standard-curve method and the newer AAC, approach. For the latter, two mRNA samples were used. One contained both the test gene and the Gapdh housekeeping gene, and the other was a "calibrator" sample. It contained the same housekeeping gene and a reference—for example, the test gene in a nontreated cell line. The C( values of all four were used to calculate the fold increase. Although the researchers noted that the AAC, method assumes 100% reaction efficiency and identical slopes for each gene analyzed conditions that may not always be me(- this approach permitted higher throughput. In addition the results obtained were in agreement with the standard-curve method and with published Northern blot analyses (Anal Biochem 1999 270 41-49)
Analytical Chemistry News & &eatures, August 1, 1999 5 1 5 A
