Primer design

The MDR1 regions (promoter and intron 1) of interest are based on the GenBank sequence X58723 (see Table 10.2 and Figure 10.1). For PCR and related work (e.g. DNA sequencing), the primers are designed to meet six criteria: a) amplicon size <400 bp, b) primer length >17 and <24 nucleotides, c) primer to target melting temperature (Tm) >67°C and <71°C (by % GC method), d) Tm for hairpin or homodimer <40°C, e) no apparent heterodimer formation for paired (forward and reverse) primers, and f) no close homology to other known sequences in the human genome. The first four criteria are evaluated using the OligoTech program (Oligos Etc, Wilsonville, Oregon), while the Entrez BLAST search is deemed sufficient in confirming the correct annealing sites for each primer. The oligonucelotides used are purchased from MWG Biotech, Inc. (High Point, North Carolina), where full-length products are recovered following standard HPSF purification, equivalent to reverse phase, high-performance liquid chromatography (HPLC). Each lyophilized oligonucleotide is resus-pended in TE80 (10 mM Tris-HCl, pH 8.0, 2 mM EDTA) as 0.5 mM stock. A

Exon 1

4 4--


Figure 10.1

Schematic view (not drawn to exact scale) of qPCR-based analyses of MDR1 gene, which has several BstU I sites that can be analyzed for methylation status. Solid and broken arrows indicate forward and reverse primers, respectively (see Table 2). When BstU I-digested genomic DNA is used as the template, PCR 2 (308 bp) defines the total MDR1 copy number in each sample, while PCR 1 (140 bp) and PCR 3 (283 bp) qunatifies the methylated DNA copies resistant to digestion by BstU I. The ratio of undigested DNA to total DNA reflects the status of CpG methylation within the BstU I sites.

1:25 dilution in distilled, PCR-grade water makes the 20 yM working solution for PCR.

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