Real Time PCR Advanced Biotechnology Lab I Florida Atlantic University April 2, 2008
Introduction We wish to compare the expression levels of our gene under study (Drosophila MsrA) for two different treatment conditions: with and without ecdysone. Since the expression levels of most genes are very low as a percentage of total RNA, the first step that we must perform is an amplification of the messenger RNA (mrna). Polymerase chain reaction is a commonlyused technique for amplification of DNA molecules.
Polymerase chain reaction (PCR) Invented in 1983 by Kary Mullis, who won the Nobel Prize in Chemistry in 1993 for the technique Amplifies a target region of DNA complimentary to a set of primers Depends on the use of a heat-stable DNA polymerase from the hot-springs bacterium Thermus Aquaticus The reaction proceeds in three steps: Denaturation at 95 C Primer annealing at approx. 60 C Elongation at 72 C
Polymerase chain reaction (cont.) See the animation at: http://www.sumanasinc.com/webcontent/animations/content/pcr.html
Reverse transcription: RNA to DNA If we want to amplify mrna, there is an initial step needed before PCR: reverse transcription of the RNA to DNA.
Gene expression studies with traditional (endpoint) PCR Once we amplify a target mrna, there will be a sufficient quantity of cdna to visualize on a gel. By amplifying the transcripts from different genes, or the same gene under different treatment conditions, we can compare the relative expression levels by observing the intensities of the bands on a gel.
Shortcomings of endpoint PCR for gene expression studies Low precision Low sensitivity Limited dynamic range (< 100) Poor size resolution Size-based discrimination only Not automated Results are not expressed as numbers Ethidium bromide stain is not very quantitative Post-PCR processing steps required
Introduction to real-time PCR Rather than waiting for the end of a defined number of PCR cycles to measure the amount of DNA, we can monitor the growth of DNA in real time. Doing this requires an assay for quantifying the amount of DNA and an instrument which can function as both a thermal cycler and a recording device. Linear scale Log scale
SYBR Green I dye SYBR Green I is a double-stranded, DNAbinding fluorescent dye. The level of fluorescence from the dye increases greatly once it binds to DNA. SYBR Green I has an absorbance maximum at 497 nm and an emission maximum at 520 nm. The main shorcoming of SYBR Green I is that it binds to all dsdna, regardless of whether or not the DNA contains the target sequence. It will therefore bind to primer dimers, secondary structures and undesired amplicons, thus interfering with accurate quantification.
Sequence-specific probes for real-time PCR To overcome the problems associated with the nonspecific binding of SYBR Green I, a variety of sequence-specific probes have been developed. These probes contain a fluorophore bound to a singlestranded oligonucleotide which is complementary to a specific sequence in the target gene. Initially, the fluorescence of the probe is blocked due to close proximity of the fluorophore and a quenching moiety. Fluorescent resonance energy transfer (FRET) occurs between the fluorophore (R) and a nearby quenching moiety. A blackhole quencher (BHQ), also called a dark quencher, emits no light after absorbing energy from the fluorophore.
TaqMan probes The TaqMan probe remains quenched until hydrolyzed by the 5'-3' exonuclease activity of the Taq polymerase. Once the probe is hydrolyzed, the free fluorophore is no longer in close proximity to the quencher. Free probe (quenched) Annealed probe (quenched) Hydrolyzed probe (fluoresces) See the animation at http://www.scanelis.com/webpages.aspx?rid=679
LUX (Light Upon extension) primers With TaqMan probes, you still need a separate set of primers to amplify your target. LUX detection technology uses one primer labeled with a single fluorophore and a corresponding unlabeled primer, both custom-synthesized according to the target of interest. LUX Primers are designed with a fluorophore near the 3' end in a hairpin structure, a configuration with intrinsic quenching capability, making a separate quenching moiety unnecessary. When the primer becomes incorporated into the doublestranded PCR product, the fluorophore is de-quenched, resulting in a significant increase in fluorescent signal.
Lux primers (cont.) With LUX primers, one primer forms a self-quenching hairpin structure while the other primer is of conventional design. Extension of the hairpin structure activates fluorescence of the reporter moiety. As additional amplicon molecules are formed, the strength of the fluorescence signal grows.
Additional probe types There are a number of additional probe types offered by various suppliers: Molecular beacons Scorpion probes Amplifluor primers Plexor primers You must insure that the probe/primer is compatible with your equipment. Of particular concern are the excitation and emission wavelengths. For further information, see the animation at: http://biosearchtech.com/download/flash_guides/formats_explained.html
Opticon 2 instrument
Comparison of real-time with endpoint PCR The real-time instrument gives a cycle-by-cycle assay of the amount of your target amplicon. By using the appropriate fluorophores, up to 8 targets can be monitored in one reaction (this is called multiplexing). Small differences in the abundance of amplicons can be detected, unlike endpoint PCR.