Lecture 8 - polymerase chain reaction

Question 1

PCR

Answer 1

Polymerase chain reaction

Highly specific and can be used to amplify, or copy specific DNA targets from a mixture of DNA molecules

Question 2

Taq DNA polymerase

Answer 2

From thermophilic bacteria

Thermostable enzyme so can repeat PCR cycling without the need to add fresh DNA polymerase after each cycle

Question 3

Basic requirements for PCR

Answer 3

DNA sequence of target region must be known
Template DNA for amplification
Primers- short oligonucleotides that will bind to a target sequence and prime DNA synthesis
Thermostable DNA polymerase which is not inactivated by heating to 95 degrees
dNTPs- deoxynucleoside triphosphate for each base
Thermal cycler- a machine that can be programmed to carry out heating and cooling of samples over a number of cycles

Question 4

How many primers are required?

Answer 4

2 to flank the target sequence

Question 5

Describe primer requirements

Answer 5

18-25 nucleotides in length
Similar Tm (50-65 degrees)
Similar GC content (which determines Tm)
GC clamp at the 3’ end to aid polymerisation initiation
Minimal secondary structure (avoids primers annealing to each other)
Minimal repeats
Minimal runs of a single base (no more than 4)

Question 6

What is the main potential primer problem?

And what would the outcomes be?

Answer 6

Need to avoid self- complementarity or complementarity to another primer

Hairpins
Self-pairing
Inter-primer pairing

Question 7

What happens if a primer annealing overhangs?

Answer 7

It could prime DNA synthesis to form primer dimers which are products formed by primers attaching to each other.

Question 8

How do you prevent primer dimers?

Answer 8

Use less primer in the reaction 
Adjust the magnesium chloride concentration 
Use more template DNA
Adjust annealing temperature 
Redesign primers

Question 9

Describe primer dimer formation

Answer 9

In the annealing stage two primers, which have complementary parts, anneal with each other
Taq polymerase then elongates the strands to form complementary dsDNA. This is a primer dimer
A primer dimer can be recognised in an electrophoresis when you ding 2 bands of DNA when you only expect 1. The lower band is the primer Dimer.

Question 10

What is seen with multiple cycles of PCR?

Answer 10

Exponential multiplication
Target DNA becomes isolated and duplicated
Increased risk and possibility of mutations

Question 11

What is DNA, to be amplified, mixed with?

Answer 11

deoxyribonucleotides
thermostable polymerase
DNA primers

Question 12

Step 1 of PCR

Answer 12

DENATURATION
90-95 degrees
The double stranded DNA breaks apart to single strands allowing access for Taq polymerase and primers

Question 13

Step 2 of PCR

Answer 13

Annealing
55-65 degrees
Oligonucleotide primers bind to the regions next to the target gene which is being amplified. Primers are at higher comcentrations than the template target so they preferentially anneal to the template rather than the two strands re-annealing

Question 14

Step 3 of PCR

Answer 14

Extension
68-72 degrees
optimum temperature for DNA polymerase. Taq polymerase will fall off when it reaches the end of the template

Question 15

How do you confirm that the PCR has amplified the desired product and is the correct size?

Answer 15

Load onto an agarose gel
Hyper-ladder which contains DNA molecules of known size is loaded onto the gel
Larger molecules move more slowly
DNA is visualised by staining with ethidium bromide that fluoresces when exposed to UV light

Question 16

What are the different variations of PCR?

Answer 16

Reverse transcriptase PCR
Real time or quantitative PCR
Assembly PCR or polymerase cycling assembly 
Allele specific PCR
Methylation specific PCR

Question 17

Reverse transcriptase PCR

Answer 17

Makes a complementary DNA copy of RNA by reverse transcription followed by PCR. Can be used to determine quantities of mRNA or genome copies of RNA viruses

Question 18

Real time or quantitative PCR

Answer 18

A variation of RT-CR where the accumulation of PCR products is monitored through each cycle of the PCR
Provides accurate quantification of mRNA levels

Question 19

(Polymerase cycling) Assemble PCR

Answer 19

Overlapping primers used to assemble into large chunks of DNA
Can be used to make synthetic genes or genomes

Question 20

Allele specific PCR

Answer 20

Primers are designed that will amplify specific alleles of a gene

Question 21

Methylation specific PCR

Answer 21

Distinguishes between methylated and non-methylated DNA

Question 22

How can PCR be used as a tool in genetic fingerprinting

Answer 22

DNA profiling can be used to identify individuals

Small samples of DNA can be isolated from a crime scene to be compared with DNA from suspects or with a DNA database

Question 23

PCR & molecular archaeology

Answer 23

Ancient DNA can be isolated from archaeological specimens. DNA tends to degrade gradually even when maintained in freezing conditions (permafrost)

Question 24

Advantages of PCR

Answer 24

Sensitive, specific, rapid identification

Question 25

Why is there no product generated until the 3rd PCR cycle?

Answer 25

After cycle 1 you have a copy of the amplified single stranded DNA with either the forward or reverse promer at each end and each are extended beyond the other primer position.
At cycle 2 you have extended sections but now the opposing primer can bind.
At cycle 3 you begin to make the blunt ended target that has each primer at the 5’ end of each strand of the dsDNA product.