Major Service Items

Detection System: LightCycler 1.5 (Roche)  Primer & probe design Quantitation assay (SYBER Green, Universal Probe Library & hybridization probe system) Melting curve PCR efficiency calibration
Our Experience
Between Run: CV < 2%     Within Run: CV < 1 %     PCR Efficiency = 1.99 (As Figure)

The real-time PCR system is based on the detection and quantitation of a fluorescent reporter (Lee, 1993; Livak, 1995).  Advantages are:

?         Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in the exponential growth phase

?         An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated

?         The cleaved probe provides a permanent record amplification of an amplicon

?         Requirement of 1000-fold less RNA than conventional assays

?             No-post PCR processing (no electrophoretical separation of amplified DNA)

?              Detection is capable down to a 2-fold change.

References

l             Lee et al. (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res, 21, 3761-3766.

l             Livak et al. (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl, 4, 357-362.