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Real-Time Quantification of Genomic DNA Using DyNAzyme II DNA Polymerase and SYBR Green I Dye

en I assays require only the two primers needed for amplificationneither of them labeled.

DyNAzyme II DNA polymerase provides superior results in a wide variety of PCR applications. This polymerase, isolated from Thermus brockianus, demonstrates better thermal stability than Taq DNA polymerase, and the ability to maintain optimal activity over a broad range of reaction conditions. DyNAzyme II is also inherently more resistant than Taq to the inhibitory effects of SGI. In this study, we present real-time quantitative PCR results indicating the sensitivity and precision of assays incorporating DyNAzyme II DNA polymerase and SGI for quantitation of starting copy number using both lambda and human genomic DNA templates.


Methods
DyNAzyme II DNA polymerase was from Finnzymes (F-503L). SGI and lambda DNA were from Molecular Probes (S-7567, P-7589); human genomic DNA was from Sigma (D-3160). Reaction components were assembled in low-profile microplates (MJ Research

MLL-9651) or strip tubes (MJ Research TLS-0851) and sealed with ultra-clear strip caps (MJ Research TCS-0803). Volumes of individual components and final reaction concentrations are listed in Table 1.

SGI reagents are typically obtained at high concentrations (e.g., 10,000X). We define a 10X SGI solution as one giving 0.40.01 O.D. when measured at 495nm. SGI was diluted to a 10X working stock with 0.1X TE buffer.

The following primer set was used in PCR reactions with lambda DNA to generate a 100bp amplicon. The sequences for the primers were: forward primer, 5-GCA-AGT-ATC-GTT-TCC-ACC-GT-3, and reverse primer, 5-TTA-TAA-GTC-TAA-TGA-AGA-CAA-ATC-CC-3

The -actin primers used for amplification of human g
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