NDLI: Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR

Content Provider	PubMed Central
Author	Giglio, Steven Monis, Paul T. Saint, Christopher P.
Copyright Year	2003
Abstract	SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.
Related Links	http://dx.doi.org/10.1093/nar/gng135
Ending Page	136
Starting Page	136
File Format	PDF
ISSN	13624962
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	22
Volume Number	31
Language	English
Publisher	Oxford University Press
Publisher Date	2003-11-15
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Genetics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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