Spermine-mediated improvement of cycling probe reaction.
Cycling probe technology (CPT) represents a simple method for detection of DNA target sequences. Cycling probe technology utilizes a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target. Probe cleavage in the presence or absence of target generates CPT product or background, respectively. Addition of non-homologous DNA into the CPT reaction affects the background and CPT product. Low amounts of human DNA (4-40 ng) result in high background while higher amounts (40-400 ng) inhibit the reaction. The simultaneous addition of spermine and EGTA into the CPT reaction containing human DNA resulted in a significant release of the inhibition and a reduction of background. The presence of spermine alone caused an increase of probe cleavage whereas addition of EGTA increased the specificity of the CPT. A possible mechanism by which spermine could lead to this improvement of CPT has been proposed. Using a membrane-binding assay, the authors demonstrated that human DNA competes with the probe for binding to RNase H. Furthermore, by using a DNA-agarose column, it has been shown that such RNase H-DNA binding can be disrupted by spermine. Within the CPT reaction, similar spermine-mediated displacement of RNase H from human DNA could lead to an improved CPT efficiency.
Modrusan
Z
Z
ID Biomedical, Burnaby, Canada.
Bekkaoui
F
F
Duck
P
P
D064127:Aurora Kinases; D015342:DNA Probes / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004533:Egtazic Acid / Q000378:metabolism; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D015347:RNA Probes / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013096:Spermine / Q000378:metabolism*
D015342:DNA Probes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D015347:RNA Probes; D013096:Spermine; D004533:Egtazic Acid; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D016914:Ribonuclease H