Novel sulfotransferases cloned by RT-PCR: real proteins or PCR artifacts?
During studies designed to subclone human phenol sulfotransferase (STP and STM) sequences for use in heterologous E. coli-based expression systems, we designed two oligonucleotide primers that would allow for the simultaneous PCR amplification of expression cassettes containing the coding regions of the STP1, STP2 and STM cDNAs. Following total RNA isolation from human liver, reverse transcription of cDNA, PCR amplification under standard conditions, plasmid subcloning and restriction analysis to select for suitable ST recombinants, we recovered plasmids containing inserts corresponding to STP1, STP2 and STM. However, ten additional, closely related but apparently novel ST sequences were also isolated. Alignments of the three known ST sequences (and one published allelic variant) with these new clones revealed that each one appears to be a PCR-generated modular chimera possessing a combination of DNA segments derived from STP1, STP2 and STM. This observation should serve as an alert to the potential pitfalls of using PCR techniques for the cloning of highly related genes and their cDNA products, especially when PCR primer design allows for the amplification of multiple products in a single reaction.
Gaedigk
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Division of Clinical Pharmacology and Toxicology, Research Institute, The Hospital for Sick Children, Toronto, Ont., Canada.
Lekas
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Berchuk
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Grant
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D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA; D015238:Sulfotransferases