Adaption of SYBR Green-based reagent kit for real-time PCR quantitation of GC-rich DNA.
In the mammalian genome, approximately 50% of all genes are controlled by promoters with high GC contents. Analyzing the epigenetic mechanisms regulating their expression is difficult. Hence, we examined a method for stable quantification of such GC-rich DNA sequences. Quantification of DNA during real-time PCR is often based on reagent kits containing the fluorescent dye SYBR Green. However, these ready-made kits may not be suitable for amplifying DNA samples with a high GC content (>70%). DNA segments with eccentric GC contents are frequently found in proximal promoter areas, and their quantification may be necessary in chromatin accessibility by real-time polymerase chain reaction or chromatin immunoprecipitation analyses of epigenetic mechanisms of gene regulation. We therefore optimized the SYBR Green I FastStart reaction system by supplementing the system with dimethyl sulfoxide, betaine, and increased DNA polymerase content. Here, we describe the development of the assay and demonstrate its effectiveness for two different DNA templates, showing that these modifications allow for the reliable amplification and quantification of DNA with GC contents exceeding >70% using the LightCycler instrument.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
G J
GJ
Chang
Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany.
H M
HM
Seyfert
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
X Z
XZ
Shen
D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes; D020862:GC Rich Sequence; D008099:Liver / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I; D004247:DNA