Real Time Pcr

Front 1

How does real time quantitative PCR (qPCR) differ from traditional (endpoint) PCR?

Back 1

Endpoint PCR - Detection and quantification of the amplified sequence are performed at the end of the rxn, after the last PCR cycle.

qPCR - DNA quantity is measured after each cycle via fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of amplicons generated

Front 2

List the advantages of qPCR compared to endpoint PCR (4).

Back 2

1. Ability to monitor the progress of the PCR rxn in real time.

2. Ability to prescisely measure the amount of amplicon at each cycle, which allows highly accurate quantification of the amount of starting material present in the sample.

3. Increased dynamic range of detection.

4. Amplification and detection occur in a single tube, eliminating post-PCR manipulations.

Front 3

Name and describe the main steps of qPCR.

Back 3

1. Denaturation - incubation of the sample at high temperatures (typically 95 C) causes dsDNA denaturation and looseing of ssDNA secondary structure.

2. Annealing - Temperature is lower to a few degrees below the melting temperature of the primers, allowing them to bind DNA.

3. Extension - Temperature is set to 70 - 72 C, which is optimal for DNA polymerase to extend the primers.

Front 4

Describe two-step qPCR.

Back 4

1. Reverse transcription of total RNA or poly(A) RNA to cDNA.

2. Use of cDNA in qPCR.

Front 5

Describe one-step qPCR.

Back 5

Combines first-strand cDNA synthesis and qPCR in one tube. Requires gene-specific primers, as opposed to the oligo(dT) or random primers which can be used in two-step PCR.

Front 6

What is the optimum [MgCl2] for qPCR?

Back 6

Typically 3 mM, but can range from 3 - 6 mM.

Front 7

What is the optimal amplicon length for qPCR?

Back 7

50 - 150 bp; longer products do not amplify as efficiently.

Front 8

List the optimal characteristics of qPCR primers (7).

Back 8

-Length: 18 - 24 bp

-Secondary structure: None

-GC content: ~50%

-Avoid stretches of homopolymer sequences

-Pairs should have a Tm within 1 C

-If possible, 3' end should be GC rich to enhance annealing at the site of extension

- concentration between 50 - 500 nM for each primer (200 nM of each primer is typically effective)

Front 9

Define baseline.

Back 9

Baseline refers to the signal level during the initial cycles of PCR, usually cycles 3 - 15, when there is little change in fluorescent signal.

Front 10

How should baseline be determined?

Back 10

Baseline should account for enough cycles to eliminate background, but should not include the cycles in which the amplification signal begins to rise above background. When comparing samples, baseline should be the same for each.

Front 11

Define threshold.

Back 11

Threshold is the level of signal that reflects a statistically significant increase over the calculated baseline signal. Typically set at 10 times the standard deviation of the fluorescent value of the baseline, but can be set at any point in the exponential phase of PCR.

Front 12

Define cycle threshold (Ct).

Back 12

The cycle number at which the fluorescent signal crosses the threshold. Ct is inversely related to the starting amount of target.

Front 13

Define efficiency.

Back 13

Ideally, a PCR reaction has an efficiency of 100%, meaning that the quantity of product doubles after each thermal cycle. In practice, a good rxn should have an efficiency of 90 - 110%

Front 14

What is a melting (dissociation) curve and what can it tell us?

Back 14

-A melting curve charts the change in fluorescence observed when dsDNA with incorporated dye molecules dissociates into ssDNA as the temperature of the rxn is raised.

-Melting curves can be used to check for primer dimers and artifacts. Since Tm is affected by many factors, different PCR products can be distinguished by their melting characteristics.

Front 15

How does a 5'-nuclease (TaqMan) assay work?

Back 15

The TaqMan probe consists of an reporter and quencher molecule attached by an oligonucleotide. In solution, the dyes are in close proximity, allowing FRET to occur. This is the state of the dye at the beginning of the assay. During PCR, the primers and probe anneal to the target, and DNA pol extends the primer upstream of the probe. If the probe is bound to the correct target sequence, it will be cleaved by the 5' nuclease activity of the polymerase, release the reporter from the quencher and allowing fluorescence to occur.

Front 16

What is FRET?

Back 16

FRET, or fluorescence resonance energy transfer, occurs when two dyes in close proximity exhibit different energy levels. When the high-energy dye is excited, energy is transferred to the low-energy dye, thus causing the high-energy dye to be "quenched".

Front 17

Name and describe the two types of TaqMan probe.

Back 17

1. Non-minor groove binding (MGB) - use TAMRA as a quencher; probes must be longer than primers, which decreases specificity

2. MGB - possess a minor groove binding protein on their 3' end; allows probes to be shorter than primers, increasing specificity.

Front 18

How does a SYBR Green assay work?

Back 18

SYBR Green is a fluorescent DNA-binding dye that binds to the minor groove of any dsDNA. DNA-bound dye emits a much higher signal than unbound dye.

Front 19

What are the functions of a passive reference dye, such as ROX?

Back 19

1. Normalize for non-PCR related fluctuations in fluorescence (e.g., caused by variation in pipetting).

2. Normalize for fluctuations in fluorescence resulting from machine "noise".

3. Compensate for variations in instrument excitation and detection.

4. Provide a stable baseline for multiplex real-time PCR and qRT-PCR.

Front 20

What is the function of Uracil N-glycosylase (UNG)?

Back 20

UNG is used to reduce or prevent carryover contamination betwen PCR reactions by preventing the amplification of DNA from previous reactions.

Front 21

What is multiplex real-time PCR?

Back 21

The detection of two or more genetic sequences in the same reaction.