cDNA amplification using one-sided (anchored) PCR.
This unit presents a modification of PCR, called anchored PCR, that allows amplification of full-length mRNA when only a small amount of sequence information lying within the mRNA is available. Both the original and reamplifications use an oligo(dT) primer complementary either to the poly(A) tail of the mature mRNA [when amplifying downstream (3') to the known sequence] or to an enzymatically synthesized homopolymer tail added to the cDNA following first strand synthesis [when amplifying upstream (5') to the known sequence]. The two rounds of PCR amplification result in a single product that can be sequenced directly or cloned into an appropriate vector for further analysis.
Dorit
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Yale University, New Haven, Connecticut, USA.
Ohara
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D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D011061:Poly A / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D011061:Poly A