Nucleic Acid Quantitation

	Core Director: Samuel Rabkin, Ph.D. Email: rabkin@helix.mgh.harvard.edu Phone: 617-726-6817
	Technician: Luba Zagachin Email: lzagachin@partners.org Phone: 617-726-9587
Core Functions: This Core offers quantitative measurement of nucleic acid (DNA or RNA) levels using real-time PCR. This includes determination of the titer of helper-free HSV vectors, genotyping (transgenic mice) and analysis of RNA or DNA levels in experimental samples. The heart of the Core is the real-time quantitative PCR machine (ABI Prism 7000 Sequence Detector System), software for primer selection and data analysis, and automated nucleic acid purification and processing (ABI Prism 6100 Nucleic Acid PrepStation). Real-time quantitative PCR provides improved sensitivity, accuracy, speed, and throughput over standard PCR methods. The ABI Prism 7000 is a new instrument that replaces the ABI 7700, which has been a standard for real-time PCR. It has a detection range of over 5 logs in a 96 well format. Detection is based on TaqMan fluorescent probes with 5' reporter and 3' quencher labels, such that synthesis from flanking primers leads to cleavage of the 5' reporter dye and detectable fluorescence. TaqMan probes can be synthesized with different color dyes permitting the analysis of multiple sequences in a single PCR reaction, so that each reaction can contain an internal control. The TaqMan probes can also be used for allelic discrimination assays. SYBR green can be used in place of the TaqMan probes to detect total PCR product.