Rapid amplification of complementary DNA from small amounts of unfractionated RNA.
We have combined, in a rapid and straightforward manner, the synthesis and subsequent amplification of individual cDNA sequences from microgram quantities of unfractionated total RNA. Taq1 polymerase, a thermostable DNA polymerase, and Moloney murine leukemia virus (MMLV) reverse transcriptase share similar buffer conditions; these reactions can be performed sequentially, in a single tube, without the need for purification or changes of buffer after the synthesis of cDNA. In this way, nonspecific losses of material are minimized and the required number of cells is reduced. Cell numbers, particularly from human tissues, can be limiting; the requirement for only small amounts of unfractionated RNA makes possible the isolation and characterization of cDNAs from biological materials available in limited quantities. As a demonstration system, we report the rapid synthesis and amplification of cDNA sequences corresponding to the first exon of human immunoglobulin E (IgE). MMLV reverse transcriptase is used with specific (i.e., IgE) or generic (i.e., oligo-[dT(12-18)]) oligomers to prime first strand cDNA synthesis from unfractionated RNA isolated from a human myeloma line, U-266. The necessary primers, deoxynucleotides and Taq1 polymerase, required for second strand cDNA synthesis and the subsequent logarithmic amplification process, are then added to the reaction mixture. This technique provides a useful means of characterizing expressed and processed gene transcripts.
Doherty
P J
PJ
Department of Immunology and Rheumatology, Hospital for Sick Children, Toronto, Ontario, Canada.
Huesca-Contreras
M
M
Dosch
H M
HM
Pan
S
S
D004247:DNA / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D006801:Humans; D012313:RNA / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D019914:Taq Polymerase
D012313:RNA; D004247:DNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase