Sequential PCR amplification was performed using a BigDye(r) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA), comprising DNA polymerase, a mixture of
deoxyribonucleotide triphosphates, and fluorochrome labeled dideoxynucleotide triphosphate.
Deoxyribonucleotides are all made from a ribonucleotide precursor by the action of ribonucleotide reductase (RNR), a tetramer of two subunits, R1 and R2 (Figure 2(a)).
[4] Nonstandard abbreviations: qPCR, quantitative PCR; Cq, quantitation cycle; HRM, high-resolution melting; dNTP,
deoxyribonucleotide triphosphate.
Under physiological conditions, RR maintains stable
deoxyribonucleotide production, which is essential for DNA synthesis; accordingly, inhibition of RR activity eliminates this balance.
(28) Briefly, template DNA (6-ng genomic DNA or external PCR products) was amplified using a HotStarTaq Plus DNA polymerase kit (Qiagen), a standard protocol (0.2 mM of each primer, 160 mM
deoxyribonucleotide triphosphates, 2 U of enzyme), and cycling conditions.
About 50 L volume amplification reaction mixture containing 10 mM Tris-HCl (pH 8.4) 50 mM KCl 1 mM MgCl2 0.5 M each primer200 M each
deoxyribonucleotide triphosphate 0.5 U ofTaq DNA polymerase and 2 g of total DNA isolated from blood or 150 ng of DNA from the positive control was used in an Eppendorf (Mastercycler gradient Eppendorf AG) thermal cycler.
The essential components in a standard single target PCR contain target DNA, two oligonucleotide primers, a DNA polymerase,
deoxyribonucleotide triphosphates (dNTPs), MgCl2, KCl, and Tris-HCl buffer.
ubiquitum gp60 gene was amplified by nested PCR in a total volume of 50 mL, containing 1 mL of DNA (primary PCR) or 2 mL of the primary PCR product (secondary PCR), primers at a concentration of 0.25 [micro]M (Ubi-18S-F1 and Ubi-18S-R1) or 0.5 [micro]M (Ubi-18S-F2 and Ubi-18S-R2), 0.2 [micro]M
deoxyribonucleotide triphosphate mix (Promega, Madison, WI, USA), 3 [micro]M Mg[Cl.sub.2] (Promega), 1 x GeneAmp PCR buffer (Applied Biosystems, Foster City, CA, USA), and 1.25 U of Taq DNA polymerase (Promega).
Polymerase chain reaction (PCR) was performed using primers and
deoxyribonucleotide precursors provided by manufacturer and subsequent hybridization was done using the Twin-Cubator (Hain Lifescience) according to manufacturer's recommendations.
The PCR reactions included 3mM Mg[Cl.sub.2], 0.5 [micro]M of each primer, 2.5 [micro]L of Ex Taq buffer, 0.2 mM of each
deoxyribonucleotide, 0.5 U of Ex Taq DNA polymerase (TAKARA), 1 [micro]L of DNA sample, and double-distilled water to a total volume of 25 [micro]L.
The cells lacked enzymes for
deoxyribonucleotide production, so it is likely that the cellular urancestor itself did not harbor a DNA genome.
In addition, disturbance of mitochondrial function affects
deoxyribonucleotide triphosphate (dNTP) balance, which in turn can result in genomic instability.
