Polymerase Chain Reaction 2

Was able to sequence the Coronavirus
DNA samples to be sequenced and quantified are sent here since not all laboratories can perform these procedures.

The Philippine Genome Center

Receives samples that are systematically processed and prepared for extraction.

Sample Preparation and Extraction Room

In big laboratories, there should be a designated area for sample preparation and extraction to ___.

avoid contamination

If PCR is exposed to nuclease, it will ___.

denature

Where the nucleic acids are amplified and purified.

PCR and quantification room

PCR and quantification room houses equipment for ___.

nucleic acid quantification and gel visualization

STEPS IN POLYMERASE CHAIN REACTION

- DENATURATION
- ANNEALING
- ELONGATION or EXTENSION

Denaturation temperature

90 - 96°C

Annealing temperature

50 - 70°C

Elongation or Extension temperature

68 - 75°C

Temperature is raised to ___.

separate the DNA molecule into two single strands

is used to denature the DNA strand

Heat

Where amplification starts.

DENATURATION

- Primers will hybridize to the DNA template.
- Temperature is decreased below the melting point of the primers (3 - 5°C) to prevent them from melting.

ANNEALING

Nucleotides have a certain technique that was also used by Kary Mullis, where ___ were incorporated.

fluorescent dyes

Examples of fluorescent dyes are:

ethidium bromide
acridine orange
SYBR green

The more it fluoresces, the more the ___.

template DNA increases in number

DNA template is replicated as the DNA polymerase extends the hybridized primers in both strands of the DNA template.

ELONGATION or EXTENSION

What differentiates the Klenow fragment from TAQ DNA or Pfu DNA Polymerase

- If we still use the Klenow fragment, the enzymes will run out during the Denaturation stage because it is not heat stable.
- The latter's enzymes are heat stable and can withstand the temperature changes in the PCR process.

TYPES OF PCR

A. CONVENTIONAL PCR
B. REAL-TIME PCR
C. REVERSE TRANSCRIPTASE PCR

Traditional PCR
Qualitative; endpoint detection

CONVENTIONAL PCR

only the presence or absence

Qualitative

results are read at the end of the entire run

Endpoint

Conventional PCR requires detection of amplified DNA region through methods such as ___

Agarose Gel Electrophoresis

Conventional PCR Disadvantages:

Low sensitivity
Non-automated
Size-based discrimination

Aka qPRC (Quantitative PCR)
Detects the accumulation of pCR products real time as it occurs.

REAL-TIME PCR

If a lot is amplified → ___
If only little to none are amplified → ___

positive
negative

In qPCR, data is collected during ___

exponential growth

qPCR can determine amplification in ___, in comparison to conventional PCR where it is measured at the ___.

real time
end point

intercalating agent, mutagenic

Ethidium bromide

sits in the minor groove of DNA, non-mutagenic

SYBR Green

Measured fluorescence is proportional to the amount of ___.

amplicons (amplified DNA)

Advantage of qPCR

Allows detection of the initial amount of template DNA.

Identifies whether contaminants or unwanted DNA are being amplified during amplification.

NEGATIVE CONTROL

If negative control becomes positive:

sample preparation was wrong

- Make sure that the reagents work.
- This allows one to know exactly at which step you went wrong.

POSITIVE CONTROL

If positive control becomes negative:

somewhere in the master mix when preparing the reagents something went wrong

REAL-TIME PCR AMPLIFICATION CURVE

LAG PHASE / INITIATION PHASE
EXPONENTIAL PHASE
PLATEAU PHASE

Flatline - no amplification occurs yet

LAG PHASE / INITIATION PHASE

Length of lag phase is ___ with the starting template you have

inversely proportional

The more DNA template you have, the ___ the lag phase

shorter

The less DNA template you have, the ___ the lag phase

longer

PCR products begin to amplify.
Increase in fluorescence
When a lot of your target DNA sequence is present, we can already see the fluorescence in the earlier cycle.

EXPONENTIAL PHASE

Quantification Cycle. Through there the amplicons can be quantified.

Cq value

Threshold Cycle

Ct value

tells you how much DNA is in your sample.

Cq / Ct value

PCR product that is amplified

Amplicons

The certain point where the numbers stop increasing exponentially.
Reaction components have run out which explains the plateau.

PLATEAU PHASE

Used when your template is RNA (e.g. HIV, HCV, COVID-19)

REVERSE TRANSCRIPTASE PCR

Reverse transcriptase PCR uses the enzyme ___ which synthesizes ___

Reverse Transcriptase (RT)
cDNA

RNA viruses like COVID-19 latch onto ___ present in the nose, lungs, and alveoli.

ace receptors

Examples of RNA viruses

Hepatitis C
Human Immunodeficiency Virus (HIV)
COVID 19