Pyrosequencing is more accurate at detecting a difference between low numbers of mononucleotide bases, such as one dATP
versus 2, as opposed to the difference between 8 and 9 dATPs
The researchers found that increased production of the enzyme Ribonucleotide Reductase increased the concentration of dATP
within heart cells approximately tenfold, and even though this level was still less than one to two percent of the cell's total pool of ATP, the increase led to a sustained improvement in heart muscle function, with the genetically engineered hearts contracting more quickly and with greater force.
After these PCR products have been labeled with [alpha]-[[sup.33]P] dATP
, they would be displayed by autoradiography as a ladder on a sequencing gel.
The second reaction consisted of (1x) PCR buffer with 1.5 mM Mg[Cl.sub.2] 200 [micro]M of dNTP containing each of dATP
, dCTP, dGTP, and dTTP, 1 U of Taq DNA Polymerase, the PCR primers and 3 ([micro]of 1:10 diluted first reaction DNA.
The 1x buffer contained the following: 1.5 mmol/L Mg[Cl.sub.2] 0.4 mmol/L each dATP
, dGTP, dTTP; 2.0 [micro]L 3,000 Ci/mmol [alpha]-[sup.32]P-dCTP (MP Bioscience, Buxton, UK); and 1.25 U Taq polymerase in a 50.
Five microliters of selected 1:10 diluted cell culture isolates was added to the reaction mixture which contained the following: 10 [micro]L of 5X reaction buffer; 2 mM magnesium sulfate; 250 [micro]M each of dATP
, dCTP, dGTP, and dTTP; 0.4 [micro]M of each EV13_VP1sense and EV13_VP1 anti-primers; 1 U of avian myeloblastosis virus RT; and 1 U of thermus flavus DNA polymerase and Rnase-free distilled water to a final volume of 50 uL.
In patients homozygous for ADA deficiency, very little dAdo accumulates during cell turnover, being reconverted to dATP
by adenosine kinase (EC 126.96.36.199) and deoxyadenosine kinase (EC 188.8.131.52) (1).
The amplification reactions were performed in a volume of 50 [micro]L containing 25 ng high-molecular-weight genomic DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl, 0.2 mM each of the dATP
, dCTP, dGTP and dTTP (Roche Diagnostics GmbH, Mannheim, Mannheim, Germany), 3 mM magnesium acetate, 30 ng primer, and 2.5 U Amplitaq DNA polymerase (Applied Biosystems, Foster City, CA).
One microgram of adenosine deaminase (ADA) was then added, and the reaction was incubated at 37 [degrees]C for 10 min to remove residual dATP
and then heated to 90 [degrees]C for 3 min to inactivate ADA activity.
Reaction volume was increased to 40 [micro]L containing (final concentrations) 50 mM Tris-HC1 (pH 8.3.), 75 mM KCI, 8 mM Mg[Cl.sub.2], 10 mM DTT, 1 mM each of dATP
, dCTP, dGTP, and dTTP, 20 units of RNase inhibitor, and 200 units of M-MLV reverse transcriptase.
Genomic DNA template (2.5 [micro]L; equivalent to 5% of the extract from a 3-mm-diameter blood spot) was amplified in the following: 25 [micro]L of 20 mM Tris-Cl, pH 8.4; 50 mM KCl; 1.5 mM Mg[Cl.sub.2]; 200 [micro]M each of dATP
, dCTP, and dGTP; 190 [micro]mol/L of dTTP; 10 [micro]mol/L of digoxigenin (DIG)-11-dUTP (Roche); and 0.75 U of Platinum Taq DNA polymerase (Life Technologies).