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M9460671.TXT
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1994-06-25
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Document 0671
DOCN M9460671
TI Design and use of signature primers to detect carry-over of amplified
material.
DT 9408
AU Abbott LZ; Spicer T; Bryz-Gornia V; Kwok S; Sninsky J; Poiesz B;
Department of Medicine, SUNY Health Science Center, Syracuse; 13210.
SO J Virol Methods. 1994 Jan;46(1):51-9. Unique Identifier : AIDSLINE
MED/94230651
AB Signature primer pairs designed for use with the polymerase chain
reaction have been developed which can determine if a positive result
originated from the intended target nucleic acid or from so-called
carry-over contamination of previously amplified DNA. The 3' ends of
each signature primer, SK339/341, SSK110/111, and SSK58/59 contain a
viral specific sequence complementary to regions of either HIV-1, HTLV-I
and II respectively. The 5' ends of each primer contain a non-human,
non-viral (NHNV) signature sequence including restriction endonuclease
sites for subsequent cloning. A fourth set of primers, SK338/340,
consist solely of these NHNV sequences and are designed to anneal to any
product previously amplified by the viral-specific signature primers.
These primers were tested against their corresponding positive and
negative DNA targets, to determine their specificity and sensitivity. As
expected, the viral-specific signature primers detected the retroviral
infected samples while no detectable amplification occurred in negative
DNA controls. Primers SK338/340 did not amplify any viral positive or
negative template DNA's. Samples spiked with amplified material
generated from the viral-specific signature primers could be
specifically amplified by the NHNV primers SK338/340. Primers SK338/340
were determined to be more sensitive than the viral-specific signature
primers, ensuring the detection of extremely low amounts of carryover.
This strategy may be useful in developing other retroviral or
non-retroviral primers with a built-in signature sequence that can
differentiate false positives from true positives in a subsequent
confirmatory test.
DE *Artifacts Base Sequence Cell Line Comparative Study *DNA Primers
DNA, Viral/*ISOLATION & PURIF Equipment Contamination False Positive
Reactions HIV-1/GENETICS/*ISOLATION & PURIF HTLV-I/GENETICS/*ISOLATION
& PURIF HTLV-II/GENETICS/*ISOLATION & PURIF Molecular Sequence Data
Polymerase Chain Reaction/*METHODS Proviruses/GENETICS/*ISOLATION &
PURIF Sensitivity and Specificity Support, U.S. Gov't, P.H.S. JOURNAL
ARTICLE
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).