Can you obtain a better representation of complete cDNAs if you use the random primers or oligo dT primers in the first-strand cDNA synthesis step for cDNA library construction?

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First-strand cDNA synthesis is most commonly primed using oligo(dT) or modifications of this sequence (such as primer-adapters) that bind to the poly(A) tail of mRNA. This priming method offers two major advantages: only poly(A)+ RNA is copied, and most cDNA clones begin at the 3’ terminus of the mRNA.

At the same time, oligo(dT) priming has certain limitations: some cDNA clones may not be full length (due to RNA secondary structure or pausing by reverse transcriptase) and poly(A)- mRNA cannot be copied.

An alternative method is to use random hexamers, which, in theory, are capable of binding and priming throughout virtually any RNA template. Random hexamers, which may be used either by themselves or in combination with oligo(dT), have been instrumental in producing cDNAs containing more 5’ information than those primed with oligo(dT) alone. In addition, random hexamers can be used to generate cDNA libraries from poly(A)- mRNA and single-stranded viral RNAs.

Answer Id: E3288

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