PCR Crash Course Part I

What is PCR?

• Polymerase Chain Reaction

• Polymerase is an enzyme that facilitates the synthesis of a variety of polymers, specifically DNA or RNA.

Analysis of PCR

Multiple ways to analyze PCR, both qualitative and quantitative.

DNA Replication Steps

1. Unzipping: separating nucleic acid pairs, usually facilitated by helicases (enzymes/motor proteins)

2. DNA polymerase gathers nucleic acid base pairs to synthesize new DNA

3. The final product: two (hopefully) identical DNA strands

Performing PCR

The 4 main ingredients for PCR:

• polymerase

• nucleotides

• template DNA

• PCR Primers

Polymerase

enzyme that facilitates DNA synthesis

Nucleotides

base monomers of DNA

Template DNA

the sample DNA strand that contains a target sequence (the thing you want to replicate)

PCR Primers

short sequence of nucleotides (20 bp in length see “oglionucleotide”)

Performing PCR Primers: What do they do? 

PCR Primers bind to template DNA and define the region that will be replicated and amplified

Where do they come from?

Primers are designed specifically for thetarget region of the tem

Why are they called primers?

Primers by definition, initiate actions. In this case, PCR primers are required to initiate PCR.

3 Main steps of PCR

1. Denaturation

2. Annealing

3. Extension

Denaturation

process by which hydrogen bonds are broken between nucleotide base pairs of double stranded DNA (dsDNA) to create two single strands (95C)

Annealing

Primers bind to the target region of the template DNA (See amplicon) (55) 

Extension

DNA polymerase extends primers by binding free nuceltoide base pairs (72)

PCR chart

1. initial denaturation (98C)

2. denaturation (98C)

3. primer annealing (61.7C)

4. extension (72C

5. final extension (72C)

6. hold (4C)

Gel electrophoresis

technique in which fragments of DNA are pulled through a gel matrix by an electric current, seperating the fragments by size/weight/bp#.

DNA ladder

is used as reference for comparison of band locations of tested samples

In the case of the figure, which one matches with the control?

Sample 1 would be considered “positive” for the target DNA, as it matches with the positive control.

Is analysis of PCR gel electrophoresis qualitative or quantitative? 

Qualitative

Quantitative PCR (qPCR) vs PCR

The main difference between PCR and qPCR is that qPCR is a real-time method.

• In other words, you can monitor DNA amplification as it is happening.

• Quantifying amplification requires the addition of fluorescent dyes or probes that bind to target DNA.

• During amplification, these dyes or probes emit energy at different wavelengths when they are excited by a laser.

What is probe?

Probes are another type of oligonucleotide with reporter dye and a quencher

What is a quencher?

absorbs flouresnce given off by the reporter dye before the probe is degraded

What is a reporter dye?

gives off fluorescence to be detected adter the probe is degraded (fluorophore) 

Extension phase of qPCR

DNA polymerase cleav

After probe is cleaved in qPCR

light fluorescent signal is emitted by the reporter and is detected by qPCR instrument

In qPCR how is amplification quantified?

• By fluorescence output from cleaved probes

• As amplification continues, fluoresence output increase

4 main phases of amplication

• lag

• exponential

• linear

• plateau phases

Ct or Cq

• The value by which detection begins is called the cycle quantification

• in qPCR, fluorescence is measured against the number of amplification cycles required for detection.

Can be PCR be used to detect DNA?

No