Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems.
In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used. Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems. Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems. The effect remained unchanged despite increasing the PCR cycles to 40. However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost. In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C). It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations. It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile.
Nath
K
K
Long Island University, Department of Biology, Brookville, New York, NY 11548-1300, USA. knath@liu.edu
Sarosy
J W
JW
Hahn
J
J
Di Como
C J
CJ
D052160:Benzothiazoles; D004271:DNA, Fungal / Q000737:chemistry / Q000187:drug effects; D003959:Diamines; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis / Q000379:methods; D004996:Ethidium / Q000737:chemistry* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000737:chemistry*; D015636:Magnesium Chloride / Q000737:chemistry; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D013194:Staining and Labeling / Q000379:methods; D015003:Yeasts / Q000235:genetics
D052160:Benzothiazoles; D004271:DNA, Fungal; D003959:Diamines; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009930:Organic Chemicals; D011804:Quinolines; D015636:Magnesium Chloride; C098022:SYBR Green I; D004996:Ethidium