Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase.
Inadequate yields of PCR product and the generation of nonspecific PCR products can complicate genotyping studies, particularly when the DNA template is of inferior quality and/or has a low-copy number. In this study, the ability of AmpliTaq Gold DNA Polymerase to enhance the specificity and yield of amplification was evaluated in a quadruplex short tandem repeat (STR) system in which a nonspecific PCR product and poor yield had been previously observed with AmpliTaq DNA Polymerase usage. Because AmpliTaq Gold is inactive until heated during the PCR before thermal cycling, effects similar to those achieved with "hot-start" PCR were attained in a fast, simple and practical fashion. A significant enhancement in yield at the four STR loci and improved balance of alleles resulted with the use of AmpliTaq Gold. Furthermore, a non-specific PCR product, the result of mispriming, was effectively eliminated. The consistency of quality results was improved, thereby promoting successful typing of suboptimal DNA samples and enhancing the accuracy of genotyping. Since PCR product yield is elevated with AmpliTaq Gold usage, and consistent performance and low background are achieved with higher amounts of AmpliTaq Gold compared with AmpliTaq, AmpliTaq Gold can be used to augment measures taken to counteract the effects of some PCR/Taq DNA polymerase inhibitors, such as those found in blood and some forensic specimens. Studies showed that pH affects either the activity or the activation of the polymerase. AmpliTaq Gold was found to be compatible with pH 8.3 buffers, such as GeneAmp PCR Buffer and AmpFlSTR PCR Reaction Mix but not compatible with pH 9.0 buffers, such as GenePrint STR 10 x Buffer (however, conditions for the usage of AmpliTaq Gold with the GenePrint CTTv system are provided). AmpliTaq Gold is useful for the development and optimization of multiplex amplification systems, particularly those in which the primers are not well designed and/or the reaction conditions are not optimal. Finally, because AmpliTaq Gold is initially inactive, preparation of reactions at ambient temperature and automation of the PCR are facilitated. Therefore throughput can be expanded significantly with the use of AmpliTaq Gold DNA Polymerase.
Moretti
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T
FBI Academy, Quantico, VA, USA.
Koons
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B
Budowle
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B
D002021:Buffers; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D015894:Genome, Human; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020014:K562 Cells; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*; D013696:Temperature; D013698:Templates, Genetic
D002021:Buffers; D017931:DNA Primers; D004247:DNA; D019914:Taq Polymerase