Objectives . This study investigated the substrate properties of modified derivatives triphosphates purine and pyrimidine deoxynucleosides (5-propynyl-2’-deoxyuridine-5’-triphosphate, 5-propynyl2’-deoxycytidine-5’-triphosphate, 5-methyl-2’-deoxycytidine-5’-triphosphate, N6-methyl-2’-deoxyadenosine-5’-triphosphate) during their simultaneous incorporation in enzymatic reactions (polymerase chain ...

DNA polymerase from Thermus aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols. A simplified method is described here for isolating the recombinant Taq enzyme after overproduction in Escherichia coli. Purification requires 8 to 10 h and entails heat treating and clearing the E. coli lysate, followed by precipitatio...

The polymerase chain reaction (PCR) and dideoxy DNA sequencing are frequently used techniques of molecular biology which utilise DNA polymerases. The high temperatures required for PCR necessitate a thermostable enzyme for DNA amplification, and DNA polymerase derived from the thermophilic microorganism, Thermus aquaticus, is used most commonly. The high optimal polymerisation temperature of th...

We previously described in a paper published in BIOS an undergraduate lab activity involving the gene cloning, expression, and purification of Thermus aquaticus (Taq) DNA polymerase, an enzyme used in the polymerase chain reaction (PCR). Based on the large number of requests for biological materials and questions about the protocols this paper invoked, we explored methods to simplify the protei...

A multiplex PCR assay consisting of 13 Rapidly Mutating Y STR loci called RM-Yplex was developed. Platinum® Taq DNA polymerase was used to amplify the 13 Y STR loci in a single reaction in approximately 2.5 hours . In order to shorten the process with reliable results, several DNA polymerases were tested with the multiplex. Phusion® Flash High Fidelity and Platinum® Taq DNA polymerases were inv...

Polymerase chain reaction (PCR) is an in vitro technique to synthesize large quantities of a given DNA molecule that separates the DNA into two complementary strands, uses a peltier heat pump to quickly heat and cool the DNA and uses the Taq polymerase for the synthesis of DNA. Taq is a bacterium that lives by volcanic sulfer jets at the bottom of the ocean where the temperature is very high. F...

As the sole renal Na,K-ATPase isozyme, the a, Na,K-ATPase accounts for all active transport of Na throughout the nephron. This role in renal Na reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)—>thymine (T) transversion, resulting in the substitution of glutamine with leucine and assoc...