Improved-primer extension preamplification (I-PEP)
We have followed the I-PEP protocol as described in Dietmaier et al. (Dietmaier W. Multiple mutationanalyses in single tumor cells with improved whole gnomeamplification. Am J Pathol 1999;154:83†95.), which is essentially a modification ofthe original PEP amplification method developed by Zhanget al. (Zhang L. Wholegenome amplification from a single cell: Implications for geneticanalysis. Proc Natl Acad Sci USA 1992;89:5847†51.)
PCR was conducted in a 30 ml reaction containing 0.01†50 ng of genomic DNA, 500 pmol of 15-base totally degenerate random primer, 200 mM each dNTPs, 2.5 mM MgCl2, 1 ml of 10X PCR buffer, 1.5 ml gelatin (1 mg/ml), and 2.1 units of high fidelity Taq DNA polymerase (Expand High Fidelity PCR System, Roche Diagnostics).
Samples were amplified in a 9600 Perkin-Elmer PCR thermal cycler with the following cycling conditions: after a pre-denaturation step of 2 min at 94 ℃, 50 cycles were conducted, each consisting of 1 min at 94 ℃, 2 min at 37 ℃, 3 min of ramp(37†55 ℃), 4 min at 55 ℃, 30 s at 68 ℃. This was followed by a final extension step of 5 min at 68 ℃.
Amplification of Human Genomic DNA by MDA.
Human genomicDNA (300 ng to 0.03 ng, as indicated) was placed into 0.2-ml tubes in a total volume of 100 ul containing 37 mM Tris_HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 5 mM (NH4)2SO4, 1 mM dNTPs, 50 uM exonuclease-resistant hexamer, 1 unit/ml of yeast pyrophosphatase, and 800 units/ml ɸ29 DNA polymerase. Radioactively labeled ɑ-[32P] dCTP, approximately 240 cpm/pmol total dCTP, was added as indicated. Reactions were incubated for 18 h at 30°C and terminated by heating to 65°C for 3 min. Acid-precipitable radioactive deoxyribonucleotide was determined with glass fiber filters to quantitate product yield. A template heat denaturation step was included or omitted, as indicated. For heat treatment the DNA template was incubated in 50 ul at 95°C for 3 min and chilled to 4°C in a PCR System...