The PCR was performed in 20 [micro]l reactions, which included of a 1:10 or 1:100 DNA dilution, 1 U REDTaq DNA polymerase (Sigma-Aldrich Company, Milan, Italy), 2 [micro]l REDTaq buffer supplemented with 1.7 [micro]l of 22 mM Mg[Cl.sub.2] for a final concentration of 3.0 mM, 10 mM deoxyribonucleotide
triphosphates, 0.5% Bovine Serum Albumin (BSA), and 0.5 [micro]M of each of the 4 primers (M1F, M1R, M2F, and M2R) [18, 19].
PDRN is obtained from the sperm of raised trout as a mixture of deoxyribonucleotide
polymers with a chain length of 50-2000 base pairs.
PDRN contains a mixture of deoxyribonucleotide
polymers with chain lengths ranging from 50 to 2000 bp and is a source of purine and pyrimidine deoxynucleosides/ deoxyribonucleotides
and bases .
RNAse inhibitor, onestep enzyme reverse transcriptase and deoxyribonucleotide
triphosphates (dNTPs) were used during the PCR.
For sequencing fluorescent di deoxyribonucleotide
terminators were applied in an automated sequencer ABI 3700 (Applied Bio Systems, USA).
A ready-to-use 2xPCR mixture from AandA Biotechnology (Poland) was used, comprised of a recombinant Taq DNA polymerase (0.1 U/l), PCR buffer optimized by the manufacturer, magnesium chloride (4.0 mM), a mixture of deoxyribonucleotide
triphosphates (0.5 mM of each dNTP), a red dye, gel loading buffer (enabling direct application of the reaction mixture on an agarose gel) and deionized water.
triphosphates (dNTPs, 10 mM stock) were obtained from QIAGEN, Inc.
Caption: FIGURE 2: Deoxyribonucleotide
by ribonucleotide reductase.
1 [micro]g of RNA was reverse-transcribed (RT) using MuLV reverse transcriptase, 1 mM deoxyribonucleotide
triphosphate (dNTP), and 0.5 [micro]g/[micro]l oligo ([dT.sub.12-18]).
The total RNA was added to reverse transcriptase buffer containing 25 mmol/L MgCl[sub]2, 10 mmol/L deoxyribonucleotide
triphosphates, 50 pmol/[micro]l random 9 mers, 40 U/[micro]l RNase inhibitor, and 5 U/[micro]l avian myeloblastosis virus reverse transcriptase to prepare a final total volume of 25 [micro]l.
Since the ORF94 is located between the rr1 and rr2, this enzyme catalysis the formation of deoxyribonucleotide
precursors, which are involved in DNA replication process.
After varying cycle numbers, the amount of polymerase, and the amount of deoxyribonucleotide
triphosphates (dNTPs), it was found that restricting PCR amplification was best done by using a dNTP concentration of 3-12 [macro]mol/L.