Synthesis of Double-Stranded Complementary DNA from Poly(A)+mRNA
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The use of avian myeloblastosis virus reverse transcriptase (AMV RTase) to produce DNA copies of mRNA templates is a common and well-documented method ( 1 – 3 ). Briefly, the method involves synthesis of a complementary DNA strand to the mRNA from a short double-stranded region, usually provided by using an oligo(dT) primer on poly(A) + RNA. The enzyme does not always produce full length transcripts, but all the complementary strands are finished off with a short hairpin loop. This provides a ready-made primer for second strand synthesis, useful whether this is to be performed by more reverse transcriptase or by E. coli DNA polymerase 1 (pol 1). An idealized picture is shown in Fig. 1 . Before the double-stranded cDNA (ds cDNA) copy can be cloned it is necessary to remove this hairpin loop using the single-strand specific nuclease S1.
Fig. 1. Stages in the production of double-stranded cDNA from poly(A) + mRNA. The original RNA is represented by a solid line, while the cDNA is represented by a dashed line. Note that this diagram is not intended as an accurate representation of the enzymatic processes involved, but as a general guide to the principles of cDNA synthesis.
