Mismatch cleavage detects pathogenic microorganisms.
When a DNA probe hybridizes a DNA target and generates a G/A mismatch in the probe-target DNA heteroduplex, the mismatch enzyme, mutY, will cut the A base at the site of the mismatch. This specific cleavage at the mismatched A on the known probe will reveal the complementary DNA sequences of the targets. This study shows mismatch cleavage assays identify the complementary sequence of cryptic plasmid target in the extract of chlamydia infected cells in culture. In addition, the specific cleavage at a single base permits differentiation of two sequences with one base difference. This was shown in differentiating the subtypes of human immunodeficiency virus (HIV) type 1. The addition of amines in the assays increases the sensitivity by freeing the target for recycling. The combined assay system of high sensitivity and demonstrated specificity allows further evaluation for direct identification of pathogenic microorganisms in patient samples.
Hsu
I C
IC
Department of Pathology, School of Medicine and Toxicology Program, University of Maryland, Baltimore, USA. ihsu@umaryland.edu
Shih
W K
WK
Lovchik
J
J
Wang
Y H
YH
Kong
D
D
D000085:Acetates / Q000494:pharmacology; D000818:Animals; D020137:Base Pair Mismatch; D002460:Cell Line / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification*; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D045647:DNA Glycosylases; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry; D005240:Feasibility Studies; D015752:Genes, env; D019302:HIV Envelope Protein gp160 / Q000235:genetics*; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D020180:Heteroduplex Analysis; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D000085:Acetates; D004269:DNA, Bacterial; D018076:DNA, Complementary; D004279:DNA, Viral; D019302:HIV Envelope Protein gp160; D015345:Oligonucleotide Probes; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; C018824:ammonium acetate