PCR - AMPLIFICATION OF NUCLEIC ACIDS

Polymerase Chain Reaction

It is an in vitro enzymatic reaction to amplify a defined DNA region.

1. Denaturation

2. Annealing

3. Extension

3 steps in PCR

• Denaturation - 92 to 95°C

• Annealing - 50 to 60°C

• Extension - 72 to 74°C

What are the temperature?

• Denaturation

• Annealing

• Extension

16S rRNA

PCR targets what gene in bacteria?

Mitochondrial genes (mtDNA)

PCR targets what gene in plants?

• Paternity testing

• Maternity testing

• Rape cases

• Forensic cases

Human identity-based testing examples

Long noncoding regions

What is the target in human identity-based testing?

Viral gene

What is the target when determining the viral load of a virus-infected patient?

True

True or False.

Viral genes can be DNA or RNA.

• N-gene

• ORF

The target genes in SARS-COV

• Genetic testing

• Pathogen detection

• Drug Development

• Crop modification

• Forensic analysis

• Sequencing

Applications of PCR

• Template DNA

• Oligonucleotide primers

• DNA Polymerase

• dNTPS

• Buffer with Magnesium Chloride (MgCl2)

What are the components of a conventional PCR?

DNA template

This is the fragment or portion of DNA that has to be amplified.

Sense strand

It is also known as the coding strand.

Antisense strand

It is also known as the complementary strand.

Oligonucleotide primers

These are short pieces of DNA or oligonucleotides that prime the polymerase reaction.

Primers

They drive the success of the annealing process. It is a crucial part of the PCR.

Fail

If there is no primers in the PCR, it will?

1. 5’ and 3’, flank

2. 15-30 BP, 50% guanine and cytosine

3. Above 50-60°C

4. Complementary, prime dimers

5. Secondary structures, self annealing

Considerations in Choosing Primers

1. Primers must be complementary to the ___ and ___ regions of the DNA template that ___ the sequence to be amplified.

2. Primers should be between ___ base pairs long and be comprised of ______.

3. The melting temperatures of both primers should be ___ _____.

4. Primers can’t be ___ to each other and form ___ ___.

5. Primers should not contain ___ ___, which is formed by ___ within one of the primers.

• <15 base pairs

• Bind to nontarget sequences

• It is indicative of too short primers

• Repercussion

• >30 base pairs

• Too long for the primers to bind to target sequence → longer annealing step, inefficient;

• Can cause self-annealing (hairpin loop)

• It is indicative of too long primers

• Repercussion

To effectively find the target sequence:

Long noncoding regions of the DNA are sequences that are not translated into proteins and are made up of guanine and cytosine.

Why are primers 50% guanine and cytosine?

50-60°C

The ideal temperature where the primers can attach to their target sequence.

False.

A self-annealing primer or inter-primer annealing will fail PCR.

True or False.

If a primer self-anneals or anneals with another primers, it is still viable for PCR as long as there are no primer dimers.

DNA Polymerase

This is the main enzyme in the DNA elongation.

DNA Polymerase

It catalyzes the synthesis of DNA by assembling the nucleotides and building new strands.

DNA Polymerase

These are enzymes that create DNA molecules by assembling nucleotides.

Nucleotides

The building blocks of DNA

Taq polymerase

The enzyme most commonly used in PCR

Thermus aquaticus

Taq polymerase comes from a thermostable bacterium called?

To survive high temperatures

Why is Taq polymerase used in PCR?

• Has its own repair mechanism

• High correctness rate (fidelity)

Why is thermus aquaticus used? What is its advantages?

dNTPs

These are the building blocks of DNA and will comprise the base pairs in the growing strands.

• Buffers

  • Manganese

  • Magnesium

  • Potassium

It maintains the pH and contains important ions such as ___, ___, and ___, which serve as cofactors.

• 10X Buffer

• 25 mM MgCl2

• 10 mM dNTPs

• 50 ng/uL Forward primer

• 50 n/uL Reverse primer

• 5 U/uL DNA polymerase

• 100 ng DNA template

• PCR Grade water

The components of a Mastermix

Denaturation

It denatures dsDNA into ssDNA so that the DNA synthesis will happen. It destroys the hydrogen bonds that are holding the double helix.

Below 72 to 74°C

The optimal temperature of Taq polymerase

4°C

The temperature for the final hold step

Final hold step

It is used to balance or neutralize the temperature of the PCR machine.

Denaturation → Annealing → Extension

One whole cycle includes?

20-35 cycles

How many cycles is needed in the PCR if the user is sure that there is presence in the sample?

45 cycles

How many cycles is needed in the PCR if the user is checking if there is presence of bacteria/ virus?

2N

N = number of cycles

After a cycle finishes, the single strand produces 2 daughter strands. In the second cycle, the 2 strands will produce 2 daughter strands of its own, leading to amplification. What is the formula?

Amplicons → Gel Electrophoresis

Analysis of PCR Products steps

• Cathode (-)

• Anode (+)

Colors in the electrophoresis

• Black

• Red

Because DNA are also negatively charged

Why are the wells facing the negative charge?

• TRIS acetate EDTA

• TRIS borate EDTA

The buffer used in electrophoresis

• Different samples / analytes

• More efficient

Why is PCR modified?

Reverse-transcriptase PCR

It was developed to amplify RNA targets.

• HIV

• HBV

Reverse transcriptase comes from what viruses?

They hijack host cells (CD4) so that they will be the ones to produce DNA

Why are HIV and HBV hard to cure?

• Oligo dT

• Random hexamers / decamers

The primers in RT-PCR

Nested PCR

It consist of 2 rounds of PCR: 1 normal PCR, and the 2nd uses amplicons from 1st round.

Nested PCR

It is developed to increase both the sensitivity and specificity of PCR.

Multiplex PCR

Multiple target sequences are being detected

• Similar annealing temperatures

• Not complementary

The primers in Multiplex PCR must be?

Different in size to form distinct bands in gel electrophoresis

The amplicons in Multiplex PCR must be?

Metagenome

Multiplex PCR is used in this area of Molecular Biology where you detect all bacteria.

• Size of PCR product

• Length of Primer

• Annealing temperature

Parameters that affect PCR

Below 3 Kb

The target sequence must be what size?

• Longer processing time

• Resource intensive

The target sequence that is more than 10 Kb will cause?

• Too high: primers cannot stay annealed

• Too low: mismatch annealing

The consequences of annealing temperature

• Too high

• Too low

• Insufficient amplification. Use 20-35 cycles.

• Insufficient time for complete replication

Troubleshooting of PCR

• Too few cycles were used

• Extension time was too short

• Do not have enough time to bind

• Primers cant bind to template

Troubleshooting of PCR

• Annealing time was too short

• Annealing temp was too high

• DNA will denature; amplification efficiency is low

• DNA will be degraded

• DNA will not completely denature and amplification efficiency is low

Troubleshooting of PCR

• Denaturation temp is too low

• Denaturation time is too long

• Denaturation time is too short

qPCR

It is the technology for the quantification of nucleic acids. It detects the accumulation of amplicon after each thermal cycle in real time.

• Conventional PCR

• Quantitative PCR

Determine if Quantitative or Conventional PCR.

• Qualitative and Semiquantitative

• Qualitative and Quantitative

• Quantitative PCR

• Conventional PCR

Determine if Quantitative or Conventional PCR.

• Real time analysis

• Endpoint analysis

• Conventional PCR

• Quantitative PCR

Determine if Quantitative or Conventional PCR.

• Post PCR analysis

• No post PCR analysis

• Conventional PCR

• Quantitative PCR

Determine if Quantitative or Conventional PCR.

• Gel electrophoresis

• Probe based or intercalating dye

Amplification curve

The result produced by qPCR

Number of DNA copies or amplicons

The y axis notes the?

Number of cycles

The x axis notes the?

Sigmoidal curve (S curve)

If the DNA is present, it will be signified by a?

• Exponential Phase

• Plateau Phase

The Sigmoidal curve is separated into 2 portions, which are?

Exponential Phase

It is where you observe an increase in number of amplicons.

Plateau Phase

It is where the curve stops; fed up reagent.

Endpoint PCR

It observes PCR product only once the plateau phase is reached.

qPCR

It allows the monitoring of PCR product during exponential phase, as its doubling with every PCR cycle.

Fluorescent dye → fluoresce → dsDNA

qPCR uses a ___ dye that only ___ when bound to ___.

Cycle treshold

It is the point where the number of DNA copies / amplicons meets with the number of cycles.

Amount of fluorescence in reaction

In qPCR, it serves as a readout of how many copies of DNA have been made.

Cycle treshold

The cycle which fluorescence is first observable above background level.

Stray lights detected

The background treshold are background noises. An example of this is?

Photosensitive

(when quencher and probes are exposed to too much light, the quencher will dissociate, and the probe will fluoresce even with no DNA → false positive)

The reagents used in qPCR are?

Inversely correlated

The CT value is ___ with the initial sample.

A test to monitor patients with communicable diseases → HIV and HBV → amount of virus in the body since it is not treatable

qPCR is used for Viral load testing. What is it?

Directly linked

A reaction Ct is ___ to the starting concentration of the target sequence.

It started with twice as much target sequence

If a reaction reaches a CT earlier than other reactions, it means?

1. Integrated excitation light source

2. Fluorescence detection system or fluorimeter

3. Software that displays the recorded fluorescence data as a DNA amplification curve

qPCR instrument is a thermal cycler with?

• Lamp

• Laser

• LED (light-emitting diode)

Examples of an integrated excitation light source

• Dye-labeled probe

• Fluorophore-labeled probe

Since all real-time PCR instruments monitor sample fluorescence during thermal cycling, it is necessary to add ___ or ____ to the reaction mixture.