Published Online: 2003
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Genomic DNA was extracted from peripheral blood samples using a rapid non-enzymatic method (1). The PCR primers and the amplification parameters of D1S80 were as described by Kasai et al. (2) for DYS19 as per Kayser et al. (3) and de Knjiff et al. (4) for DYS287 (YAP) as per Hammer and Horai (5) and for DYF155S2 as per Jobling and Tyler-Smith (6). PCR amplification of all the four loci was achieved by using locus specific primers flanking the repeat region and was carried out in an eppendorf™ Master Cycler. For D1S80 and DYF155S2, the amplimers were resolved on 4% non-denaturing polyacrylamide gel followed by silver staining, whereas for DYS287 (YAP) locus, the amplimers were electrophoresed on 2% agarose gel stained with ethidium bromide. For DYS19 locus, the forward primer was labeled with flourescent CY5™ dye amidite and the amplimers were electrophoresed on 6% denaturing urea gel (7M) using ALF Express DNA Sequencer (Amersham Pharmacia Biosciences, PVT. Ltd., Uppasala, Sweden). Internal ladders were used in addition to external standards (CY5™ labeled 50-500 bp DNA ladder) for accurate size determination. The amplimers of DYS19 locus were also compared with the standards, kindly supplied by Dr. Chris Tyler-Smith from Oxford University, Oxford, UK, for confirmation.
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