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For each individual reaction, a total 20 [micro]l volume contained 2 [micro]l 10x SYBR green buffer; 1 [micro]l cDNA; 0.5 [micro]l 25 pmol/[micro]l each primer; 2.4 [micro]l (25 mM) Mg[Cl.sub.2] Solution; 1.6 [micro]l dNTP Blend (2.5 mM each dATP, dCTP, dGTP, 5.0 mM dUTP); 0.2 [micro]l (1 u/[micro]l) Amp Erase[R] UNG; 0.1 [micro]l (5 u/[micro]l) Ampli Taq Gold[TM] (Applied Biosystems, Foster City, CA, USA) and 10.7 [micro]l dd[H.sub.2]O.
In the current assay, primers immediately adjacent to codon 600 on the forward and reverse strands were extended in a ddATP and dGTP, dCTP, and dTTP mixture that gave extensions of 1 to 7 nucleotides, depending on the sequence (Fig.
The reaction contained 500 [micro]M each of dATP, dCTP, and dGTP; 250 [micro]M dTTP; 50 [micro]M Cy5-dUTP; 300 [micro]M each of the primers; and 3.5 U of the Expand-Long Template DNA polymerase (Roche) in the supplied reaction 1X buffer.
One microliter cDNA was used in competitive PCR reaction mixtures performed in total volumes of 12 [micro]L (final concentrations, including contributions from the cDNA): 12 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.9 mM Mg[Cl.sub.2]; 0.1% Triton X-100; 0.005% gelatin; 14 [micro]M each dATP, dCTP, dGTP, and dTTP; 1 [micro]Ci [[sup.35]S][Alpha]dATP (Amersham Pharmacia Biotech); 10 pmol each upstream and downstream primers (Table 1, Table 2); and 1 U AmpliTaq (Perkin-Elmer, Norwalk, CT).
PCR was performed in volumes of 50 [micro]L containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM Mg[Cl.sub.2], 100 [micro]M each of dATP, dCTP, dGTP, dTTP, 0.2 [micro]M primer, 0.001% (w/v) gelatin, 30 ng of genomic DNA, and 1 unit of Taq polymerase.
Following this, 20 [micro]L of a mixture containing 1.5 [micro]mol/L of each deoxynucleotide triphosphate (50% Cy5-labeled dCTP and dTTP; Amersham Biosciences), 2x extension buffer, 0.5 g/L bovine serum albumin, and 20 [micro]g of proteinase K was then added to initiate the extension by the polymerase and simultaneously terminate the extension by degradation of the polymerase by the protease.
A total volume of 2.5 [micro]L of the extracted DNA was amplified in a 25-[micro]L reaction mixture containing 12.5 pmol of each primer, 200 [micro]M of dATP, dCTP, dGTP, and dTTP, and 1 U of Elongase in 1X PCR buffer with 0.8 [micro]L of 25 mM MgC12 (Life Technologies, Cergy Pontoise, France).
Briefly, 50 ng of parental and [F.sub.2] DNA was used as template in a 20 [micro]L of reaction containing 1 x reaction buffer (10 mM Tris-HCL, 50 mM KCL, pH 8.3); 2.5 mM MgCl2; 2 [micro]M of each primer; 50 [micro]M each of dATP, dGTP, and dTTP; 1 [micro]M of dCTP; 1 [micro]M of [[Alpha].sup.32]P-dCTP; and 1.0 unit of Taq polymerase.
The PCR mixture contained PCR buffer [50 mM Tris-HCI, 10 mM KCI, 5 mM ([NH.sub.4])S[O.sub.4], 2 mM Mg[Cl.sub.2]], nucleotides (0.2 mM each of dATP, dCTP, and dGTP and 0.6 mM dUTP), FastStart Taq DNA polymerase as provided by the manufacturer, 0.5 [micro]M each of forward primer TLR4 S and reverse primer TLR4 A, and 0.2 [micro]M SimpleProbe oligomer Sensor 299 [A]4L, specific for A896G (TIB MOLBIOL; Table 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).
PCR reactions for the BSR3.sp1, K375.sp1, 14H13.sp1, 21E22.sp1, 21E22.sp2, 30L19.sp1, 35E22.sp1, and 98P22.sp2 markers were carried out in a 20-[micro]L reaction mixture containing 60 ng of genomic DNA, 0.5 [micro]M of each primer, 1x Gibco-BRL PCR buffer, 1.5 mM Mg[Cl.sub.2], 100 [micro]M each of dGTP, dTTP, dATP and dCTP, 0.5 U Taq Polymerase (Gibco-BRL), and 0.5x SCR dye [6% (w/v) sucrose, 100 [micro]M cresol red].
The amplification mixture contained 250 ng of genomic DNA of probands, 10 mM Tris-HCl, 2.5 mM Mg[Cl.sub.2], 50 mM KCl, 125 [micro]M each deoxynucleotide (dATP, dGTP, dCTP, and dTTP), 10 pmol of each primer, and 0.5 U of Taq polymerase in a final volume of 25 [micro]L.
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