PCR

What are five reagents needed for PCR?

1. Sample DNA

2. Taq Polymerase

3. Deoxynucleotide Triphosphatases

4. Primers

5. Buffers

6. Cofactors

What is another name for the sample DNA?

Template Strand

Why do we use Taq-Polymerases as opposed to regular Polymerases?

Taq Polymerases are not denatured at high temperatures like regular Polymerases.

Where were Taq Polymerases isolated from?

The bacterial species Thermus aquaticus

What are the two roles of cofactors?

1. Helps primer adhere to the correct site

2. Helps Taq-Polymerase work correctly

What machine is used to perform PCR?

Thermocycler

What are the three phases of a PCR reaction?

1. Denaturation

2. Annealing

3. Extension

What is the purpose of Denaturation? At what temperature does this take place?

1. To separate the two DNA strands

2. 95 degrees Celsius

Why must a high temperature be used in order to separate the strands?

Breaks the hydrogen bonds in the middle of the molecule.

What is the purpose of Annealing? What temperature does it take place at?

1. To adhere primers

2. 55 degrees Celsius

What is the average length of a primer?

18-22 nucleotides

Why is the length of a primer important?

Shorter primers anneal to the strand more easily, but it increases the chances of annealing in the wrong place.

What is a primer’s specificity?

The tendency for it to anneal in the correct location.

How does magnesium help the primer adhere?

Both the primer and template strand have a negatively charged backbone, so they would repel each other. Magnesium (Positively-charged) helps keep the primer flipped in the correct direction.

When does the buffer work?

Only after magnesium has done its job

What does the buffer do?

It stabilizes the hydrogen bonds between the template and primer strands. It destabilizes the primer if it’s bound to the incorrect spot.

What is the purpose of the Extension phase? What temperature does it take place at?

1. Taq-Polymerase builds complementary DNA strand

2. 72 degrees Celsius

What direction does Taq-Polymerase build in?

5’ → 3’ only

How does Magnesium help in the extension phase?

It forms a bond between the 3’ end of the primer and the first dNTP added

How do you calculate the number of copies?

# Copies = (# starting strands)(2)^(# Cycles) 

What kind of PCR does Covid testing use?

RT-qPCR

What extra step is included in RT-qPCR?

Since Covid has RNA as genetic material and not DNA, we will need to convert RNA to DNA using an enzyme called Reverse Trascriptase

How does quantitation in RT-qPCR happen?

Uses the amount of fluorescence to quantitate the amount of genetic material. 

What are two types of fluorescent reagents?

1. SYBR Green

2. Taqman Probe

How does SYBR Green Work?

Fluorescence increases as double-stranded DNA increases (binds to dsDNA)

How does Taqman Probe work?

1. Once the DNA gets its second strand, the Taqman sequence will break off, causing it to fluoresce. 

What are two key components of the Taqman molecule? What is their purpose?

1. Reporter (glows once molecule breaks)

2. Quencher (keeps light off when molecule is attached)

What kind of error is introduced into RT-qPCR

False negatives if there’s not enough viral material present to surpass the threshold level.