In the case of precursor amiR, the precursor sequence following the MSO sequence was labeled using Cy3-dATP and all four unlabeled deoxynucleotides
(Figure 2(b)) in an individual reaction.
Reactions of 10 or 20 [micro]L contained 10 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 3 mmol/L MgCl2, 0.3 mmol/L of each deoxynucleotide
, 1 U JumpStart Taq polymerase (all Sigma-Aldrich), 0.5X SYBR Green I (from a 10 000 X concentrate; Invitrogen), and 400 nmol/L ofeach primer (MWG-Biotech).
To synthesize complementary DNA, 1.0 [micro]g of RNA was resuspended in a 20-[micro]L final volume of the reaction buffer (50 [micro]m Tris-HCL, 75 mM KCl, 10 mM dithiothreitol, 3.0 mM Mg[Cl.sub.2], 10 mM of each deoxynucleotide
triphosphate, 1 U/[micro]L of RNase inhibitor, pH 8.3; Perkin-Elmer Cetus, Foster City, CA) containing 0.5 [micro]g oligo d(T) 12-18 primer (GIBCO BRL, Gaithersburg, MD).
The complete electropherograms of the intracellular nucleotides, deoxynucleotides
, nucleosides, and bases in RBCs from healthy individuals and ADA/SCID patients are reported in Fig.
These differ from the results (C < G < A < T) based on oligonucleotides with 3' deoxynucleotides
In all shown experiments with both standard PCR mixtures and [sup.4x]Mix, we used FastStart[R] Taq DNA polymerase (5 U/[micro]L), PCR-grade deoxynucleotides
, Mg[Cl.sub.2], and buffer purchased from Roche Applied Sciences.
(3) in 100 [micro]L of reaction mixture containing 2.5 U of Taq DNA Polymerase (Life Technologies), 1 X PCR buffer (20 mmol/L Tris-HCl pH 8.4, 50 mmol/L KCl), 200 [micro]mol/L deoxynucleotides
(Amersham Pharmacia Biotech), and 3 mmol/L [MgCl.sub.2].
The PCR mixture contained 10 mmol/L Tris-HCI (pH 8.8), 50 mmol/L KCI, 1.5 mmol/L MgCIZ, 1 mL/L Triton X-100, 200 [micro]mol/L each of the four deoxynucleotides
, 1 [micro]mol/L each of the primers, and 3 U of Dynazyme (Finnzymes) in a final volume of 50 [micro]L.
The amplification mixture contained Taq buffer diluted 1:10 as suggested by the manufacturer, deoxynucleotides
(200 [micro]mol/L each), 40 pmol of each primer, and 8 [micro]L of purified genomic DNA.
D, A complementary primer (5"-AAGGGT-3') has been annealed, and if the complementary deoxynucleotide
triphosphate (dNTP) is added (dCTP), the DNA polymerase (gray oval) will incorporate it into the nascent elongating strand at the position of the open box.
Fluorescent terminal deoxynucleotide
transferasemediated dUTP nick-end labelling (TUNEL) was performed on 5 Am-thick transverse sections of sample A.