A random-PCR method (rPCR) to construct whole cDNA library from low amounts of RNA

In contrast with random-primed RACE (1) and RNA-PCR (2) which can be done to amplify respectively specific 5'or 3'-cDNA ends, random-PCR (rPCR) can be used to amplify the whole cDNA population, from 5' to 3' ends, derived from small amounts of RNA transcripts. This quick rPCR method allows construction of whole cDNA libraries from as little as one eukaryotic cell, 100 parasites or bacteria, or 10 particles of a 9 kb retrovirus, so lowering the threshold. Libraries constructed in this manner can then be screened with specific DNA probes or antibodies. Moreover, cDNA amplified by rPCR are powerful tools for the screening of substracted libraries. The rPCR was tested here on several MS2 phage RNA dilutions, either respectively 5xlO~ , 5x l0~ 5 , 5xlO~ and 5 X 10~ /tg MS2 RNA (Serva), and on a control assay with no RNA. Using a 26 nucleotides primer containing a random hexamer at its 3' end (Universal primer-dN6; 5'-GCCGGAGCTCTGCAGAATTC-3'), the first-strand cDNA was prepared as follows. MS2 RNA was suspended in 6 /tl of distilled water, heated to 65C for 5 min, rapidly cooled on ice and reverse transcribed after addition of 0.5 /tl (20 units) RNAsin, 1.25 /tl 10xreverse transcription buffer (500 tnM Tris-HCl pH 8.3 at 43°C, 800 mM NaCl, 80 mM MgCl2, 50 mM DTT), 1.25 /tl each dNTP (10 mM), 1.5 /tl Universal primer-dN6 (0.1 /tg//tl), 2 /tl (16 units) AMV reverse transcriptase. Incubation was at 43°C for 1 h. The reaction was then boiled for 2 min and rapidly cooled on ice. For second-strand cDNA synthesis, the following components were then added; 24.25 /tl distilled water, 10 /tl 5 xKlenow buffer (kit multiprime/Pharmacia), 1.25 /tl dCTP (100 mM), 2 /tl Klenow fragment (8 units). After 30 min incubation at 37 °C, the sample was purified on a Chroma Spin-400 column (Clontech) to eliminate the excess of Universal primer-dN6. Amplification of the randomly synthetised double-strand cDN A population was then performed on a 1 /tl aliquot in presence of the Universal primer essentially as described by Cetus (3). Briefly, amplification took place in 50 /tl reaction mixtures containing double-strand cDNA in 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 0.01% gelatin, 500 /tM each dNTP, 1 /iM of Universal primer and 1.5 unit Taq polymerase (Cetus). The samples were subjected to 40 cycles of amplification, 94°C-1 min, 55°C-1 min, 72°C-3 min. Final amplification products analysed on agarose gel (Figure 1) showed DNA populations essentially distributed from 0.4 to 3 Kb with a maximum number of copies around 0.8 Kb; rPCR on total eukaryotic RNA gives routinely DNA fragments around 0.5 Kb. Care has to be taken with the Universal primer-dN6 concentration as it governs both the size of the synthesized cDNA and the efficiency of the reactions. Using 1 /tl from a 50 /J assay, as little as 10~ /tg RNA was efficiently amplified with the rPCR method 0ane d). However, less starting material can generate amplification of one or a few cDNA species that are not representative of the initial RNA population (lane f); this was supported here by the use of a pure MS2 RNA which is only 3.6 kb long. The rPCR is actually used in our laboratory in attempt to identify putative retrovirus sequences poorly represented in human culture cell supernatant. This technique should also be applicable for amplification of genomic DNA by replacing the reverse transcriptase with a Klenow fragment step.