Ligation-anchored PCR: a simple amplification technique with single-sided specificity.
A simple, efficient, and sensitive technique has been developed for amplification of cDNAs encoding molecules with 5' regions of unknown sequence. In this ligation-anchored PCR, T4 RNA ligase is used to covalently link an "anchor" oligonucleotide to first-strand cDNAs. These anchored cDNAs are then amplified by using one PCR primer specific for the anchor and another specific for a sequence within the molecule of interest. The anchor oligonucleotide has been especially designed to facilitate subsequent analysis and cloning of the resultant PCR products. This three-stage procedure does not require purification of product between steps and avoids many of the technical difficulties associated with established anchored PCR protocols. The efficacy of ligation-anchored PCR was demonstrated by amplification of a specific IgG1 cDNA; total RNA equivalent to as few as 100 cells yielded the expected PCR product.
Troutt
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Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
McHeyzer-Williams
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Pulendran
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Nossal
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GJ
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012972:Sodium Hydroxide / Q000737:chemistry; D013604:T-Phages / Q000201:enzymology
D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012972:Sodium Hydroxide; D004247:DNA; D011088:DNA Ligases