Molecule detection. CleanAmpTM Primers outperform other Hot Start Technologies Since CleanAmpTM Primers provide significant benefit relative to reactions with unmodified primers, experiments were then performed to compare the performance of CleanAmpTM Primers to that of other Hot Start technologies. In these studies, the performance of unmodified primers with one of a series of Hot Start DNA polymerases, such as a chemically modified version of Taq (2), was compared to the performance of CleanAmpTM Primers with unmodified Taq DNA polymerase. Amplicon was formed with equal or much lower yield than reactions that employed unmodified Taq DNA polymerase with Precision Primers (Figure 4A, B). Moreover, Turbo Primers and unmodified Taq DNA polymerase gave the greatest benefit, as the amplicon yield was much higher than each of the Hot Start polymerases examined. These findings are significant, as they indicate that CleanAmpTM
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Precision CleanAmpTM Primers (0.5 ), SYBR Green (.15X ), ROX (30nM), dNTPs (0.2 mM), 0-50,000 copies Lambda gDNA, 1.25 U Taq DNA polymerase, 25; reactions performed in duplicate. Thermal cycling conditions: 95 (10 min); [95 (40 sec), 57 (30 sec), 72 (1 min)] 40X.611168-24-2 site Figure 3: SYBR Green real-time PCR assay where 0-50,000 copies of Lambda gDNA were assayed using unmodified, Turbo and Precision Primers.
Hot Start Polymerases
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Primers (0.4 M), 0.2 mM dNTPs, 5 copies HIV-1 gDNA, DNA polymerases: 1.25 units of Taq, Platinum Taq, AmpliTaq Gold, HotStart-IT, DyNAzyme, and 2.5 units of Herculase, 50 mL. Thermal cycling conditions: 95 C (10 min); [95 Figure 4: polymerases with Hot Start activation C (40 sec), 56 C (30 sec), 72 C (1 min)] 35X other *denotes Comparison of CleanAmpTM Primers to commercially available Hot Start DNA polymerases. A. Endpoint PCR analysis of amplification reactions containing 5 copies of HIV-1 genomic DNA. B. Graphical representation of the relative amplicon yield, normalized to reactions containing Taq DNA polymerase plus CleanAmpTM Turbo Primers.
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be impossible to differentiate between desired amplicon formation and other off target formation. The use of Turbo Primers provides at least a ten-fold increase in detection, as
Primers and unmodified Taq DNA polymerase can be employed without compromising amplicon yield, while efficiently reducing primer dimer formation and mis-priming.24939-03-5 medchemexpress
2
CleanAmpTM Primers are compatible with other DNA polymerases Taq DNA polymerase was used as a point of reference to determine whether Precision and Turbo CleanAmpTM Primers could be employed with other DNA polymerases in endpoint PCR experiments.PMID:29763052 In the endpoint reactions, seven DNA polymerases devoid of Hot Start activation were evaluated for their ability to robustly form the desired 365 bp amplicon (Fig. 5). Each of the DNA polymerases examined was able to support efficient amplification of the DNA target. For all cases, with the exception of Deep VentTM and Tfi polymerase, the units of DNA polymerase were kept constant. Overall,
DynazymeTM II DynazymeTM II Deep Vent TM Pfu (exo-) Deep Vent TM Pfu (exo-)
365 385 bp bp amplicon
amplicon
PCR conditions: 1X PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), Primers (0.4 ), 0.2 mM dNTPs, 5 copies HIV-1 gDNA, Taq (1.25U), Pfu (2.5U), Pfu(exo-) (2.5U), DynazymeTM(1.2U), Deep VentTM(2.5U), Tth (2.5U), Tfi (20U), 50.
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