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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
1994-8-22
pubmed:abstractText
The effect of phosphorothioate bonds on the hydrolytic activity of the 5'-->3' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5' biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophilic surface in the presence of salt.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1054-9803
pubmed:author
pubmed:issnType
Print
pubmed:volume
3
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
285-91
pubmed:dateRevised
2000-12-18
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.
pubmed:affiliation
Molecular Tool, Inc., Alpha Center, Baltimore, Maryland 21224.
pubmed:publicationType
Journal Article