Table-1: Mean and standard deviation values of the clinical characteristics in control, high risk (HR) and preeclampsia group (PET) Characteristics Control Group (n=40) Age (years) 31.20 [+ or -] 5.84 BMI (kg/[m.sup.2]) 29.94 [+ or -] 6.05 Gestational Age (weeks) 31.17 [+ or -] 5.33 Haematocrit (%) 34.75 [+ or -] 4.30 Platelet Count 266.17 [+ or -] 84.83 ([10.sup.3]/[micro]l) sATP (mmHg) 113.56 [+ or -] 13.93 dATP
(mmHg) 67.66 [+ or -] 9.38 S.
However, each of these reaction mixtures contained 20 nM of the template, 10 nM of each primer 5' -TATTGATTGTGAATTA(C/G)-3', and 100 [micro]M of a single dNTP (dCTP, dGTP, dATP
, or dTTP).
Because this is done in real-time where the x-axis of the graph is time, the first 3 nucleotides dispensed (deoxythymidine triphosphate [dTTP], deoxyguanosine triphosphate [dGTP], and dCTP) do not elongate the growing strand because the first complementary nucleotide is dATP
. Thus, there are no peaks on the pyrogram for these dispensed dNTPs (abbreviated T, G, and C in the graph) because they could not be incorporated into the growing strand.
For this, they used genetic engineering to create a strain of mice whose cells produced higher-than-normal levels of an enzyme called Ribonucleotide Reductase that converts the precursor of ATP, adenosine-5'-diphosphate or ADP, to dADP, which, in turn, is rapidly converted to dATP
PCR requires DNA template, target specific primers, four dNTPs (dATP
, dTTP, dCTP, and dGTP), DNA polymerase, magnesium, and buffer.
For SSR analysis, a reaction volume of 20 [micro]l was set up containing 2.0 ul of 10 x PCR buffer [50 mM of Tris (pH 8.3), 500 mM of KCl]; 1.5 mM of Mg[Cl.sub.2]; 0.2 mM each of dATP
, dCTP, dGTP and dTTP (Fermentas, USA); 0.3 mM each of reverse and forward primer (GeneLink, USA); one units of Taq DNA polymerase (Fermentas, USA); and 25 ng of genomic DNA as a template.
The supernatant was transferred to a fresh tube, the cDNA reamplified using the same primers and similar PCR conditions except radio-labeled dATP
was not used and the dNTP concentrations were increased to 250 [micro]M.
For labeling with Cy3-dATP, 0.5 mM of each dCTP, dGTP, and dTTP and 0.08 mM dATP
(Qiagen, Hilden, Germany) were used instead of 0.5 mM dTTP.
A total of 50 of the first reaction mixture contained (1x) PCR buffer with 1.5 mM Mg[Cl.sub.2] (Invitrogen, USA), 200 [micro]M of dNTP (Invitrogen, USA) containing each of dATP
, dCTP, dGTP, dTTP the PCR primers, 2.5 U of Taq DNA Polymerase (Intron, Korea), and 1 [micro]l of DNA template.
PCR amplification was carried out with PCR primers (20 pmol/L each per 50 [micro]L reaction) (Integrated DNA Technologies, Coralville, IA, USA) (Table) and 1 [micro]L genomic DNA (extracted from overnight Luria-Bertani broth cultures according to PureGene DNA isolation kit instructions [Gentra Systems, Minneapolis, MN, USA] and dissolved in 50 [micro]L 10 mmol/L Tris, pH 8.3) in a PCR cocktail containing 1x PCR buffer, 1.5 mmol/L Mg[Cl.sub.2], 1 U Vent exo(-) polymerase from New England BioLabs (Beverly, MA, USA), and 200 [micro]mol/L each dATP
, dGTP, dTTP, and dCTP.
For each run, a master mix was prepared with 1 x SYBR Green buffer containing 5 mM Mg[Cl.sub.2]; 200 mM dATP
, dCTP, and dGTP; 400 mM dUTP; 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Courtaboeuf, France); and 300 nM of each primer: EXIIc sense primer, 5' TGA GGT CAA GGA ACA CAA GA 3', exon II specific (positions 9-28); and EXIII antisense primer, 5' ATC CAC AGG AAT CTG CCG TG 3', for exon III (positions 211-230) (Corbin et al.