Open AccessSimple and effective method for generating single-stranded DNA targets and probes
Author(s) -
Xing Tang,
Sheldon L. Morris,
John J. Langone,
Larry E. Bockstahler
Publication year - 2006
Publication title -
biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000112154
Subject(s) - microbiology and biotechnology , dna , hybridization probe , pneumocystis carinii , biology , primer (cosmetics) , dna microarray , polymerase chain reaction , mycobacterium tuberculosis , primer dimer , nucleic acid thermodynamics , gene , southern blot , molecular probe , dot blot , chemistry , gene expression , genetics , virology , tuberculosis , multiplex polymerase chain reaction , base sequence , human immunodeficiency virus (hiv) , medicine , organic chemistry , pathology , pneumocystis jirovecii
A simple and efficient PCR method was developed for generating dye- or radiolabeled single-stranded DNA targets or probes used for hybridization studies. The method involved the use of a pair of long primers with high annealing temperatures and a short, labeled primer with a low annealing temperature in a PCR consisting of two cycles at different temperatures. We used this method to generate dye Cy 5-labeled and [32P]-radiolabeled single-stranded DNA targets and probes. These labeled probes were used successfully for the microarray identification of point mutations in Mycobacterium tuberculosis genes and for the Northern blot detection of expression changes of the GATA-2 gene in Pneumocystis carinii-infected rat lungs.
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