(q)PCR, SPECIFICITY AND SENSITIVITY By Krystal Charley

POLYMERASE CHAIN REACTION (PCR) Developed in 1983 by Kary Mullis Developed in 1983 by Kary Mullis Only a little bit of target DNA is needed to amplify Only a little bit of target DNA is needed to amplify Not as specific as qPCR: Not as specific as qPCR: The amount of PCR result does not relate to starting amount The amount of PCR result does not relate to starting amount Relies on gels to stain/see bands (products): Relies on gels to stain/see bands (products): -Ethidium Bromide -Gel Red Primers: Primers:-specific-universal Can it be more specific? Have more sensitivity? Can it be more specific? Have more sensitivity?

USES FOR PCR Medical and Biological research Medical and Biological research Cloning Cloning Forensic Sciences Forensic Sciences Diagnosis of hereditary diseases Diagnosis of hereditary diseases Diagnosis of infectious diseases Diagnosis of infectious diseases Identification of fingerprints Identification of fingerprints Paternity testing Paternity testing

QUANTITATIVE PCR (qPCR) Also called Real Time PCR Also called Real Time PCR Does not require the use of gels Does not require the use of gels Two approaches to qPCR: Two approaches to qPCR: Measuring the Ct (Cycle Threshold) Measuring the Ct (Cycle Threshold) Double-stranded DNA banding dye/Fluorescent dye Double-stranded DNA banding dye/Fluorescent dye Expensive Expensive

Ct is the number of cycles required for the fluorescent signal to cross the threshold. Ct is the number of cycles required for the fluorescent signal to cross the threshold. In PCR, the area measured is the plateau (or the stationary phase) of the cycles. In PCR, the area measured is the plateau (or the stationary phase) of the cycles. In qPCR, the exponential area (log phase) is where the cycles are measured. In qPCR, the exponential area (log phase) is where the cycles are measured. Can know the amount of starting material and monitor how much DNA is made before the plateau. Can know the amount of starting material and monitor how much DNA is made before the plateau. Sensitivity is critical at this point. Sensitivity is critical at this point. CYCLE THRESHOLD

FLUORESCENT DYE This technique/usage of a probe makes qPCR very specific: This technique/usage of a probe makes qPCR very specific: Detects only the DNA sequence complementary to the probe Detects only the DNA sequence complementary to the probe A DNA-based probe is added along with primers for PCR and annealed: A DNA-based probe is added along with primers for PCR and annealed: Contains a fluorescent tag and quencher (inhibitor) at opposite ends of the probe Contains a fluorescent tag and quencher (inhibitor) at opposite ends of the probe The probe is degraded by polymerase and the fluorescent tag is separated from the quencher in the process. The probe is degraded by polymerase and the fluorescent tag is separated from the quencher in the process. This results in increasing fluorescence. This results in increasing fluorescence.

MEASUREMENT OF FLUORESCENCE

IMPORTANCE IN MEDICAL AND BIOLOGICAL RESEARCH Most commonly used to quantify viruses in the blood of patients

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