Overview of Real-Time PCR

This set of flashcards covers key terms and concepts related to real-time PCR, including processes, enzymes, and technical details.

Real-time PCR

A variation of the standard PCR technique used to quantify DNA or RNA in a sample.

Amplification

The process of increasing the number of copies of a specific DNA or RNA sequence.

Fluorescent probes

Used for quantification of amplified products in real-time PCR.

Reverse transcriptase (RT)

An enzyme that converts RNA into complementary DNA (cDNA) during RT-PCR.

Template DNA

The strand of DNA that is amplified during the PCR process.

dNTPs

Deoxynucleotide triphosphates, the building blocks of DNA used in PCR.

Taq DNA polymerase

A thermostable DNA polymerase commonly used in PCR that retains activity at high temperatures.

SYBR Green I

A dye that binds to double-stranded DNA, producing a fluorescence signal that increases as more DNA is amplified.

Threshold cycle (C)

The cycle number at which the fluorescence of the PCR amplification exceeds a threshold level.

Cyclic Polymerase Chain Reaction (PCR)

A technique used to amplify specific DNA sequences through repeated cycles of denaturation, annealing, and extension.

Hot-start enzyme

A type of enzyme that is inactive at room temperature, minimizing nonspecific product synthesis during reaction setup.

Gene-specific primers (GSPs)

Primers designed to bind to a specific gene sequence for amplification in PCR.

Quantification

The determination of the amount or concentration of DNA or RNA in a sample.

Melting temperature (Tm)

The temperature at which half of the DNA strands are in the double-helix state and half are in the 'melted' single-stranded state.

Cycle threshold (Ct) value

A measurement used in quantitative PCR to determine the starting quantity of a nucleic acid.

Mg2+ (Magnesium ions)

A cofactor required for the activity of DNA polymerase during PCR.

PCR efficiency

The effectiveness of a PCR reaction in amplifying DNA.

Endogenous 5' nuclease activity

The ability of Taq DNA polymerase to cleave probes, contributing to the generation of fluorescence.

False positives

Incorrect results indicating the presence of a target sequence when it is not actually present, often caused by nonspecific amplification.

Random primers

Primers used in the reverse transcription step that do not bind to specific sequences, allowing broad cDNA synthesis.