OVERVIEW OF PCR

Polymerase Chain Reaction

The first specific amplification method of any type. It is also the first and prototypical method for target nucleic acid amplification.

Kary Mullis

He discovered PCR in the mid-1980s, which he was granted a nobel prize for in 1993.

Polymerase Chain Reaction

It is a copy machine for DNA that revolutionized molecular biology.

False.

PCR is used to determine and confirm cellular or organismal presence by the detection of its genomic DNA or RNA (bacterial, viral, eukaryotic organisms).

Modified True or False.

PCR is used to determine and confirm cellular or organismal presence by the detection of its genomic RNA (bacterial, viral, eukaryotic organisms).

True

Modified True or False.

PCR is used to characterize cells or organisms by determining the DNA/RNA sequence of an amplified DNA/RNA target

Amplicons

These are the copies of a specific DNA sequence. It is formed after PCR.

In vitro

PCR is DNA replication ___ ___

DNA Polymerase

What is the main replication enzyme?

Base Pairing Rule (A-T, G-C)

DNA replication follows what rule?

False.

By amplifying the amount of nucleic acid materials obtained from clinical samples. Molecular techniques can effectively provide a quantitative representation of an individual's health

Modified True or False.

By amplifying the amount of nucleic acid materials obtained from clinical samples. Molecular techniques can effectively provide a qualitative representation of an individual's health

• Water

• Buffer

• DNA Template

• Thermostable Polymerase

• Primer Pair

• Magnesium (Mg2+)

• Deoxynucleotide triphosphates (dNTPs)

What are the components of PCR?

Water

It is the medium for all other components.

• Purified

• Double distilled

• Deionized

• Autoclaved

• Nuclease free

• No detectable amounts of nucleic acid

Describe the water used for PCR.

Buffer

It stabilizes DNA polymerase, DNA, and nucleotides.

DNA Template

It contains the target region of interest.

• Thermostable polymerase

• Taq polymerase

• It is used to catalyze DNA-dependent DNA synthesis.

• Give an example

Thermus aquaticus

Taq polymerase comes from what thermophilic bacteria?

Primer Pair

These are oligodeoxynucleotides that flank the region of interest.

Forward Primer Pair

A type of primer pair that locates the replication start point along the template’s sense strand.

Reverse Primer Pair

A type of primer pair that marks a second replication start point along the antisense, downstream of its opposite primer.

18-30 base nucleotides

The length of a primer pair should be?

Magnesium

It acts as a cofactor for polymerase activity. It also stabilizes the DNA double helix.

• Too little: Enzymes will not work

• Too much: DNA will become extra stable and will cause non-specific priming

What are the consequence(s) if Magnesium is:

• Too little

• Too much

dNTPs

These are the equimolar building blocks of DNA.

Pre-PCR → PCR → Post-PCR

What is the typical PCR set-up in a laboratory?

• Isolation of nucleic acid from samples

• Preparation of PCR reaction components (mastermix)

• Addition of nucleic acid template into the PCR Reaction Mastermix

Pre-PCR includes?

• PCR

• Denaturation, Annealing, Elongation

• This PCR set-up includes Thermal Cycling

• What is it composed of?

40

How many cycles can be done in thermal cycling?

Post-PCR

It includes the analysis of amplified DNA.

Pre-PCR

It is done prior to performing Molecular Biology techniques. It is the first step in PCR analysis.

• Cellular lysis

• DNA / RNA extraction

Sample treatment requires?

Heat denaturation

It refers to the high heat used to disrupt and separate DNA duplexes.

Heat

It breaks the hydrogen bonds of DNA template and separates into single strands.

94 C

DNA is heated to a minimum of (what temperature)?

Primer annealing

It refers to the attachment of primers to the template strand.

50 - 60 C

The DNA is cooled down to (what temperature)?

Individual ssDNA

DNA primers bind to ___ in annealing phase.

Primer extension

It refers to the elongation of daughter strand with DNA polymerase.

72 - 74 degrees C

What is the temperature in elongation?

• Repetitively

• Turnaround time

The PCR cycle would continue ___ in a predetermined ___.

Running the correct controls

It is essential for ensuring and maintaining the accuracy of the assay.

Positive controls

It ensures that the enzyme is active, the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately.

Negative control without DNA

It ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run.

• Contamination control

• Reagent blank

A negative control without DNA is also called?

Negative control with DNA

It lacks the target sequence (negative template control) and ensures that the primers are not annealing to nontarget sequences of DNA.

• Gel electrophoresis

• Capillary Electrophoresis

• Nucleic acid hybridization

• DNA sequencing

• Real-time quantitative PCR

In Post-PCR, what are used to analyze PCR products?

False.

Depending on the application, the size, presence, or intensity of PCR products is observed after electrophoresis.

Modified True or False.

Depending on the application, the size, presence, or intensity of PCR products is observed before electrophoresis.

• Reverse Transcriptase PCR (RT-PCR)

• Multiplex PCR

• Real-time Quantitative PCR

The modifications of traditional PCR

Reverse Transcriptase PCR

It refers to the conversion of the RNA template into its complementary DNA strand (cDNA), which is an essential step in the analysis of gene transcripts.

The cDNA is sequenced, cloned, and applied to estimate the copy number of specific genes in order to characterize and validate gene expression.

RNA virus

The enzyme reverse transcriptase comes from what organism?

Multiplex PCR

It is a powerful technique that enables amplification of two or more products in parallel in a single reaction tube. It typically employs different primer or probe pairs in the same reaction for simultaneous amplification of multiple targets.

Human Papilloma virus

What organism can be used in multiplex PCR?

Multiplex PCR

It is used widely in genotyping applications and multiple areas of DNA testing in research, forensic, and diagnostic laboratories.

Multiplex PCR

It is used for the detection of multiple pathogens using multiple primer sets in a single reaction.

Ladder

Ladder

It is used as a standard to compare the bands from the gel electrophoresis.

Real time / Quantitative PCR

In this PCR, the accumulation of amplification product is measured as the reaction progresses with product quantification after each cycle.

Real Time / Quantitative PCR

Viral load of Hepatitis B virus is acquired through?

• Early: high amount of starting template

• Late: low amount of starting template

In Real Time or Quantitative PCR:

The detectable fluorescence in earlier cycles of the amplification program indicates ____, while fluorescence in later cycles indicates ____.

Isolate DNA

Quantitative real time PCR first step

• Opaque

• Fluorescent dye can be easily degraded in transparent PCR tube due to misdirection

• What PCR tube is used for fluorescent dyes?

• Why?

Centrifugation

A procedure that can remove bubbles.

Treshold cycle (CT)

The PCR cycle at which sample fluorescence crosses the threshold is the?

Fluorescent reporter molecule

Real-time detection of PCR products is enabled by the inclusion of a ____ ____ ____ in each reaction well that yields increased fluorescence with increasing of product DNA.

Ethidium bromide

The first approach in qPCR utilized ___, which is specific to double-stranded DNA. However, due to its toxicity, it was replaced.

• SYBR Green

• Specific, robust fluorescence, reduced toxicity

• Ethidium bromide is replaced by ___. It is also specific with dsDNA.

• What is its advantage?

Carcinogen

Ethidium bromide is a known?

Probe

Its purpose is to identify one or more sequences of interest within a large amount of nucleic acid.

False.

The probe should hybridize specifically with the target DNA or RNA that is to be analyzed.

Modified True or False.

The probe can either hybridize or neutralize the target DNA or RNA that is to be analyzed.

• Taqman

• Molecular Beacons

• Scorpions

• Fluorescent Resonance Energy Transfer

What are the common probes used in qPCR?

TaqMan

It was developed from one of the first probe-based systems for quantifying cDNA by qPCR.

Molecular Beacons

It measures the accumulation of product at the annealing step in the PCR cycle. It does not elongate or extend.

Scorpions

It is a target-specific primer which are tailed at the 5' end with a sequence complementary to part of the internal primer sequence, a quencher, a stem-loop structure, and a 5' fluorophore.

Fluorescent Resonance Energy Transfer (FRET)

It utilizes two specific probes, one with a 3' fluorophore (acceptor) and the other with a 5' catalyst for the fluorescence (donor).

Acceptor

The 3’ Fluorophore in FRET

Donor

The 5’ Catalyst of fluorescence in FRET

FRET

This PCR transfers energy.

True

Modified True or False.

Real-time PCR results can either be qualitative (the presence or absence of a sequence) or quantitative (copy number).

TaqMan probe

It hybridizes to the target sequences between the PCR primer-binding sites. The probe is covalently attached to a fluorescent reporter dye (R) at the 5' end and a quencher (Q) at the 3' end.

5’ end

At what end is Reporter Dye seen?

3’ end

At what end is Quencher seen?

True

Modified True or False.

In TaqMan, when the quencher and reporter dye are together, there will be no fluorescence signal.

Reporter Dye

This contains the fluorescence signal.

TaqMan signal fluorescence

It is generated when Taq polymerase extends the primers and digests the probe and releases the reporter from the vicinity of the quencher.

Molecular Beacon Probe

It contains target-specific sequences and a short, inverted repeat (~5 bp) that hybridizes into a hairpin structure. The 5' end of the probe has a reporter dye (R), and the 3' end has a quencher dye (Q).

True

Modified True or False.

In the presence of target sequences (Molecular Beacon), hybridization of the probe will open the hairpin, moving the quencher from the reporter and allowing signal fluorescence, which doubles with every doubling of target sequence.

Scorpion

An advantage of this system is the covalent attachment of fluorescent signal to the PCR product, which is useful for further analysis, such as size assessment by capillary electrophoresis.

False.

FRET probes are separate oligomers, one covalently attached to a donor fluor (D) and one to an acceptor or reporter fluor (R). The acceptor/reporter will fluoresce only when both probes are bound next to one another on the target sequences. As more target accumulates, more probes bind, and more fluorescence is emitted.

Modified True or False.

FRET probes are separate oligomers, one covalently attached to a donor fluor (D) and one to an acceptor or reporter fluor (R). The acceptor/reporter will fluoresce only when both probes are bound next to one another on the target sequences. As more target accumulates, less probes bind, and more fluorescence is emitted.

Sigmoidal curve

True amplification displays what curve?

Exponential phase

This phase refers to the increase of DNA copies.

Plateau phase

The enzymes increased are finished and there are no bases to be added, it has reached what phase?

Baseline

Low-level signals attributed to the background or the "noise" of the reaction.

• Bubbles

• Vibrations

• Incorrect pipetting

PCR is sensitive. If there is noise, it means it is unstable. It can be caused by?

Treshold line

The minimum level of fluorescence to determine a CT value. It is above the baseline.

False.

CT is inverse to starting template amount.

Modified True or False.

CT is proportional to starting template amount.

CT value

When the fluorescence crosses the treshold line, it determines the?

Contamination

Late amplification without sigmoidal curve means?

• Sigmoidal amplification

• CT value

What are the 2 requirements to determine the result in qPCR?