O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases.
The concentrations of nest 1 and 2 were identical. Each 20-µL reaction mixture for nest 1 amplifications contained 2 ng of P. falciparum genomic DNA in the absence or presence of 5%, 10%, 15%, 20%, or 40% blood eluent (or 40% elution buffer). Two microliters of the nest 1 amplification product were used as the DNA template for each of the 20-µL amplifications.
The concentrations of nest 1 and 2 were identical. Each 20-µL reaction mixture for nest 1 amplifications contained 2 ng of P. falciparum genomic DNA in the absence or presence of 5%, 10%, 15%, 20%, or 40% blood eluent (or 40% elution buffer). Two microliters of the nest 1 amplification product were used as the DNA template for each of the 20-µL amplifications.
| KOD FX | MightyAmp | Hemo KlenTaq | Phusion Blood | KAPA Blood | BIOTAQ | Go Taq | |
|---|---|---|---|---|---|---|---|
| 5′-primer | 0.3 µM | 0.3 µM | 0.3 µM | 0.5 µM | 0.5 µM | 0.5 µM | 0.5 µM |
| 3′-primer | 0.3 µM | 0.3 µM | 0.3 µM | 0.5 µM | 0.5 µM | 0.5 µM | 0.5 µM |
| PCR buffer | 1x | 1x | 1x | 1x | ‡ ‡ denotes dNTPs and PCR buffer components were included in the DNA polymerase mixture vial. | 1x§ § denotes that the PCR buffer was supplied by SHIMADZU Corporation. | 1x |
| dNTP | 0.4 mM | * * denotes dNTPs were included in the PCR buffer. | 0.2 mM | * * denotes dNTPs were included in the PCR buffer. | ‡ ‡ denotes dNTPs and PCR buffer components were included in the DNA polymerase mixture vial. | * * denotes dNTPs were included in the PCR buffer. | 0.2 mM |
| DNA polymerase | 0.02 units | 0.025 units | (1.6 µL)† † denotes indicated volume from each supplier. | 1 unit | (10 µL)† † denotes indicated volume from each supplier. | 0.5 units | 2 units |
| KOD FX | MightyAmp | Hemo KlenTaq | Phusion Blood | KAPA Blood | BIOTAQ | Go Taq | |
| Initial denaturation | 94 °C, 2 min | 98 °C, 2 min | 95 °C, 3 min | 98 °C, 5 min | 95 °C, 5 min | 95 °C, 10 min | 95 °C, 2 min |
| Denaturation | 98 °C, 10 sec | 98 °C, 10 sec | 95 °C, 20 sec | 98 °C, 1 sec | 95 °C, 30 sec | 94 °C, 30 sec | 95 °C, 45 sec |
| Annealing | 55 °C, 30 sec | 55 °C, 15 sec | 55 °C, 30 sec | 55 °C, 5 sec | 55 °C, 30 sec | 55 °C, 1 min | 55 °C, 45 sec |
| Extension | 68 °C, 45 sec | 68 °C, 45 sec | 68 °C, 45 sec | 72 °C, 45 sec | 72 °C, 45 sec | 72 °C, 45 sec | 72 °C, 45 sec |
| Final extension | 68 °C, 7 min | 68 °C, 7 min | 68 °C, 10 min | 72 °C, 1 min | 72 °C, 10 min | 72 °C, 7 min | 72 °C, 5 min |
