PCR protocols for scanning

Any recipe for PCR can be used with the simple addition of an LCGreen™ dye at 1X concentration. If capillaries are used for amplification, 250-500 pg/ml BSA (final concentration) should be added. We routinely use 50 mM Tris, pH 8.3, 500 pg/ml BSA, 200 pM each dNTP, 1-5 mM MgCl2, 0.4 U heat-stable polymerase, 88 ng of TaqStart™ antibody (ClonTech), 0.2-0.5 pM each primer and 10-50 ng of human genomic DNA in 10 pl reactions. One pl of human genomic DNA with an absorbance of 1.0 at 260 nm provides 50 ng or 15,000 copies of DNA. Either an exonuclease-positive (Roche) or exonuclease-negative polymerase (KlenTaq1™, AB Peptides) can be used. Alternatively, dye can be added to LightCycler® FastStart DNA Master HybProbe PCR kit (Roche). It is likely that by the time of publication of this book, complete PCR master mixes including the LCGreen™ dye will be available from Idaho Technology, Roche, or both. Hot-start procedures (physical temperature, wax barrier, anti-Taq antibody, or heat-activated enzymes), although not required, are recommended. All double-stranded DNA species bind LCGreen™ dyes and affect the melting profile, and although it is often possible to 'read through' undesired amplified products, it is much easier to interpret specific, single product amplifications.

Temperature cycling protocols and optimization will not be detailed here. The goal is a pure PCR product, so rapid cycling, hot-start techniques, and a limited number of cycles (30-40) are recommended. Parameters for rapid capillary PCR have been published elsewhere (Wittwer et al., 2001), although denaturation times must be modified if heat-activated Taq is used. Rapid cycling is not required, and standard PCR on 96- or 384-well plates is the best high-throughput solution. Gradient thermal cyclers are convenient for initial optimization. Real-time monitoring is not necessary, but is convenient and aids in troubleshooting. After PCR the samples should be denatured and cooled rapidly. With capillary systems, a brief '0' sec denaturation is adequate. On plate thermocyclers, a 10-30 sec denaturation is recommended. In either case, cool the samples to below 40°C at the fastest ramp possible. The samples can be melted immediately or stored at room temperature before analysis. If plates have been covered with a film for thermal cycling, remove the film before melting. Melting rates for scanning are usually 0.3°C/sec on the HR-1™ and 0.1°C/sec on the LightScanner™.

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