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Genotyping Single Nucleotide Polymorphisms with Molecular Beacons

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Methods

Real-time monitoring of polymerase chain reaction: Molecular beacons were synthesized by Stratagene. A detailed protocol for molecular beacon design is available on Stratagenes web site at http://www.stratagene.com/index.asp?catID=17. The nucleotide sequence of the molecular beacons was as follows: SNP11A: 5-fam-gcgcagagacggAacttgctctgaaactgcgc-DABCYL-3; SNP11G: 5-fam-gcgcagagacggGacttgctctgaaactgcgc-DABCYL-3. Underlined regions identify the probe sequences, and an A or G capital letter in bold indicate the A/G polymorphism. Primers FP 5-aacttcacctccccacatca-3 and RP 5-atctggggcacagaagcatt-3 (Research Genetics) were selected from the region flanking the SNP11 in the BAC and generated a 144-bp-long amplicon. Human genomic DNAs were isolated from peripheral white blood cells, and extractions were carried out with the Super QUIK-GENE DNA isolation kit (Analytical Genetic Testing Center). Each 50-ml PCR reaction contained 100 ng of human genomic DNA, 500 nM molecular beacon A or G, 400 nM of each primer, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.4 mM dUTP, 3.5 mM MgCl2, 2.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems), and 1 U of uracil-N-glycosylase (UNG). Reactions were performed in triplicate in PE Applied Biosystems GeneAmp(r) 5700 Sequence Detection System. The PCR conditions were as follows: Cycling was preceded by 2 minutes at 50C; 10 minutes at 95C, followed by 40 cycles of 15 seconds; and 1 minute at 60C. In addition, a dissociation curve was generated after the last cycle of the PCR reaction. The thermal protocol for dissociation is defined as a hold at 95C for 15 seconds, a hold at 60C for 20 seconds, and a slow ramp (20 minutes) f
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