Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

minor groovebinder (MGB)multicomponentingmulti-reporter(multiplex) PCRNo TemplateControl (NTC)normalized reportersignal (R n )nucleotideoutlierPCR master mixpersist(ed)pure dyeOne of a class of molecules which form close atomic contacts in the deep, narrowspace formed between the two phosphate-sugar backbones in the DNA double helix.Conjugation of a minor groove binder to oligonucleotides stabilizes nucleic acidduplexes causing a dramatic increase in oligonucleotide T m . Fluorogenic probes withthe MGB attached to the 3′ end perform well in the 5′ nuclease assay. They are animprovement over unmodified probes because shorter sequences (13- to 20-mers) canbe used to obtain probes that have an optimal T m . Thus, attachment of the MGBenables the use of shorter fluorogenic probes, which results in improved mismatchdiscrimination.The analysis performed by the SDS software used to distinguish the contribution ofindividual dyes to the raw spectral data collected by the 7900HT instrument. Theoverlapping spectra from the individual dye components in a well generate thecomposite spectrum that represents a raw data fluorescent reading.A technique that uses multiple fluorogenic dye-labeled TaqMan ® probes to detect thesimultaneous amplification of two or more target nucleic acids within a DNA sample.A control reaction with all PCR components included except DNA template. After adetector is applied to a well, each detector must be assigned a task that describes itspurpose. One of the tasks is NTC and is applied to all detectors of negative controlwells containing PCR reagents but lacking samples.See R n .One of the structural components, or building blocks, of DNA and RNA. A nucleotideconsists of a base (one of four chemicals: adenine, thymine, guanine, and cytosine)plus a molecule of sugar and one of phosphoric acid.An unusually extreme value for a variable, given the statistical model in use. In theSDS software, automatic outlier removal in RQ Studies is conducted to assist in theelimination of data contamination occurring when a process or phenomenon otherthan initial sample quantity affects the measured C T .A reagent mix containing all components necessary for PCR except template DNAand primers/probes. For example, the TaqMan ® 2✕ Universal PCR Master Mixcontains AmpliTaq Gold ® DNA polymerase, AmpErase ® UNG that protects againstcarry-over contamination, Passive Reference I for signal normalization in all 5′nuclease assays, dNTPs and optimized buffer components.Stored in, and thus retrievable from, the database used by the SDS software.Pure dye data is generated from the results of a pure dye run in which the SDSsoftware collects spectral data from a set of dye standards during a 2-min. hold at60 °C. The software stores the spectral information for the pure dye standards withina calibration file located in the SDS directory. This calibration file is used forcalculating multi-component data. After the run, the software extracts eachcomponent dye spectrum from the collected data in the pure spectra run file. Becausethe age and use of the instrument components can affect the pure spectra readings,Applied Biosystems recommends updating the pure spectra data files once or twiceannually depending on instrument use.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Glossary-5DRAFTSeptember 1, 2004 11:39 am, Glossary.fm