PCR

Question 1

What is PCR?

Answer 1

Technique to make many copies of a specific DNA region in vitro

Question 2

What does PCR require?

Answer 2

Thermostable DNA polymerase Taq polymerase

DNA primers designed specifically for the DNA region of interest

Nucleotides

Template DNA

Question 3

When is PCR used?

Answer 3

DNA cloning

Medical diagnostics

Forensic analysis of DNA

Question 4

What is the goal of PCR?

Answer 4

Make enough of the target DNA region that it can be analyzed or used in some other way

DNA may be

• sent for sequencing
• visualised by gel electrophoresis
• cloned into a plasmid for further experiments

Question 5

What is Taq polymerase?

Answer 5

PCR requires DNA polymerase enzyme that makes new strands of DNA, using existing strands as templated

DNA polymerase typically used in PCR is Taq polymerase

After the heat-tolerant bacterium from which it was isolated (Thermus aquaticus)

Question 6

When is Taq polymerase most active?

Answer 6

70 degrees

Question 7

What are primers?

Answer 7

Short sequences of nucleotides that provides a starting point for DNA synthesis

Experimenter determines the region of DNA that will be copied by the primers they choose

Question 8

What is the average length of primers?

Answer 8

Around 20 nucleotides in length

Question 9

How many primers are used in a PCR reaction?

Answer 9

Two primers are used in each PCR reaction

They are designed so that they flank the target region

Given sequences that will make them bind to opposite strands of the template DNA

Just at the edges of the region to be copied

Question 10

What are the steps of PCR?

Answer 10

1. Ingredients are assembled in a tube, along with cofactors needed by the enzyme
2. Denaturation
3. Annealing
4. Extension

Question 11

What temperature does denaturation happen?

Answer 11

96°C

Question 12

What happens during denaturation?

Answer 12

Heat the reaction strongly to separate or denature the DNA strands

Provides a single-stranded template for the next step

Question 13

What temperature does annealing happen?

Answer 13

55 - 65°C

Question 14

What happens during annealing?

Answer 14

Cool the reaction

So the primers can bind to their complementary sequences

On the complementary sequences on the single-stranded template DNA

Question 15

What temperature does extension happen?

Answer 15

72°C

Question 16

What happens during extension?

Answer 16

Raise the temperature so Taq polymerase extends the primers

Synthesizes new strands of DNA

Question 17

How many times is this process repeated?

Answer 17

Cycle repeats 25-35 times in a typical PCR reaction

Question 18

How many hours does a normal PCR take?

Answer 18

2-4 hours

Question 19

What enables the rapid growth of the DNA?

Answer 19

It’s not just the original DNA that’s used as a template each time

The new DNA that’s made in one round can serve as a template in the next round of DNA synthesis

Many copies of the primers and Taq polymerase floating around in the reaction

Number of DNA molecules can roughly double in each round of cycling

Question 20

How long does annealing take?

Answer 20

Around 1 minute

Determined by the binding between the bases

A and T - weaker bonds, the melting temperature is lower

C and G - stronger bonds, the melting temperature is higher

Question 21

What is a thermocycler machine?

Answer 21

Performs PCR automatically

Question 22

How can PCR be used to diagnose UTIs?

Answer 22

All eubacteria have 16s ribosomal gene that codes for 16s ribosomal DNA

Design primers that stick to the constant region on the constant DNA that codes for the 16s gene

Metagenomic sequencing

Question 23

What is metagenomic sequences used for?

Answer 23

Strain identification

Mutation

Look for genes for antibiotic resistance

Question 24

What is the advantage of PCR?

Answer 24

Only need small amount

Question 25

What are the disadvantages of PCR?

Answer 25

Need to know the sequence you are investigating

Expensive

Takes time