PCR and it's Application Lecture 5 and 6

Question 1

Polymerase chain reaction (PCR) overview

Answer 1

• invented by Kary Mullis in 1985-87
  -working at Cetus Corporation, synthesising oligonucleotides for study of mutations in sickle cell anemia
• Problem: limited sample (human genomic DNA)
• Nobel prize in Chemistry, 1993
  -Hoffmann-La Roche bought licence off Cetus for $300 million.

Question 2

The polymerase chain reaction (PCR) allows us to…

Answer 2

amplify and isolate a DNA sequence that we are interested in.

Question 3

What principle is PCR based on?

Answer 3

It is based on the principle of natural DNA replication carried out by DNA polymerase in cells.

Question 4

Using purified/recombinant DNA polymerase, where is semi-conservative amplification of DNA carried out?

Answer 4

in a test tube.

Question 5

How are only selected parts of the DNA is amplified

Answer 5

By using primers

Question 6

Natural DNA replication starts from a short RNA primer that the DNA polymerase extends at the

Answer 6

3’ end

Question 7

Initial (historic) experimental set-up:

Answer 7

• E. Coli DNA polymerase was not heat-stable, had to be added in fresh after each denaturation step.
  Now we have heat-stable polymerases
• ‘Cycling’, i.e. change in temperature had to be done manually by moving the tubes into water baths of different temperatures

Question 8

Taq DNA polymerase properties

Answer 8

*isolated from the bacterium Thermus aquaticus, which lives in hot springs,
* heat-stable DNA polymerase, which survives the denaturation step and elongates DNA at 72oC.
* first published in 1976
* Science ‘Molecule of the Year’ 1989

Question 9

What is other heat-stable polymerase are there

Answer 9

Pfu and Vent, isolated from other thermophiles.

Question 10

What is in a Typical PCR reaction:

Answer 10

H2O
Buffer
MgCl
dNTPs
Template to be amplified
2 Primers (forward and reverse)
Taq polymerase

Question 11

Denaturation

Answer 11

• 95°C
• separation of double strands

Question 12

primer annealing

Answer 12

• 50-68°C
• depends on melting temperature of the primers

Question 13

elongation.

Answer 13

72°C

Question 14

Primer design considerations

Answer 14

• need to frame (flank) the sequence that you want to amplify.
• should have at least 15bp long complementary sequence to template
• Forward and reverse primer should have a similar annealing temperature.
• Primers should not form strong primer dimers or have extensive secondary structures.

Question 15

What does annealing temperature depend on

Answer 15

annealing temperature depends on length and GC content. Usually, annealing temperatures should be between 50 and 70 ̊C.

Question 16

what is PCR used for in Medicine

Answer 16

diagnostics for viral infections (HIV), for genetic disorders, HLA typing before transplantation

Question 17

what is PCR used for in Forensics

Answer 17

amplify DNA from tiny amounts of blood, tissue, semen, DNA fingerprinting, paternity tests

Question 18

what is PCR used for in Evolutionary studies

Answer 18

molecular phylogenetics, amplify ancient DNA, e.g. from Woolly Mammoth

Question 19

How is DNA fingerprinting by PCR based on length polymorphisms carried out

Answer 19

Analyses Microsatellite regions. With PCR primers flanking the microsatellite region, the microsatellites can be amplified and their size compared on a gel.

Question 20

what are Microsatellite regions?

Answer 20

variable number of tandem repeats, (VNTR): short sequences, e.g. CA, repeated 4-30 times

Question 21

actual DNA fingerprint analysis

Answer 21

An actual DNA fingerprint analysis would analyse several different VNTR loci to reliably differentiate individuals

Question 22

Real-time PCR

Answer 22

In a Real-time PCR, we measure the DNA amplification (the amount of DNA in the tube) in ‘real- time’ after each cycle.

Question 23

what is the aim in Quantitative Real-time PCR

Answer 23

quantify the amount of template DNA (starting material).

Question 24

what is qPCR used for

Answer 24

gene expression studies

Question 25

use of mRNA in qPCR

Answer 25

• mRNA is an indicator for gene expression.
• can only use DNA (not RNA) as a template in a PCR.
• mRNA is turned into so-called cDNA using an enzyme called Reverse Transcriptase.

Question 26

Synthesis of cDNA

Answer 26

1.Poly-dT primers are used, because they anneal to the 3’ end of nearly all eukaryotic mRNAs (due to the presence of polyA-tail)
2.Reverse Transcriptase extends that primer and reverse transcribes RNA into DNA. This gives you an RNA/DNA hybrid helix.
3. RNA strand is degraded with RNAse H and a complementary DNA strand is synthesised with DNA polymerase producing a ds DNA copy of the original mRNA.

Question 27

How does the qPCR cycler measures the amount of DNA that is present after each PCR cycle in real time

Answer 27

by using an intercalating DNA dye or a fluorescent probe

Question 28

Ct value =

Answer 28

• the value where the PCR curve crosses the threshold.
• this is the value that is used for the analysis.
• The higher the Ct (30-35), the less the mRNA detected is present.

Question 29

How do you measure the relative expression

Answer 29

∆Ct value (CT housekeeping – CT GOI).

Question 30

How do you express the change in expression level of the GOI?

Answer 30

∆∆Ct (∆Ct stimulated - ∆Ct unstimulated).

Question 31

RT-PCR for SARS-CoV-2 diagnosis

Answer 31

• extract RNA from nasopharyngeal swabs (SARS-COV-2 has an RNA genome)
• This is then converted into cDNA for use in RT-PCR
• Specific primers for SARS-COV-2 genes are used in theRT-PCR (usually ‘’multiplexed‘’, so 2-3 genes are detected at the same time)

Question 32

What are the advantages of using Northern Blotting to quantify mRNA transcript levels

Answer 32

• Can determine size of the RNA transcript and could pick up alternative transcripts
• Control house keeping gene B-actin mRNA doesn’t change during stimulation, therefore this normalises for total RNA level in sample.

Question 33

What are the disadvantages of using Northern Blotting to quantify mRNA transcript levels

Answer 33

-Technicallymore difficult
-Not as quantitative

Question 34

Microarrays allow more…

Answer 34

global gene expression analysis

Question 35

microarray process

Answer 35

• DNA oligonucleotides corresponding to specific genes, are immobilised on a small glass, silicon or nylon slide. Each field contains many copies of a single stranded DNA probe.
• mRNA is extracted from cells, converted to cDNA and labelled with a fluorescent dye.
• The chip is exposed to the labelled cDNA
• The array is scanned to measure fluorescence intensity. The location of the bound sample on the array is detected and gene expression is determined.

Question 36

what is an example of microarray applications

Answer 36

Cancer Biopsy to normal tissue sample (Stimulated samples compared to non- stimulated control.)

Question 37

benefits of RNAseq

Answer 37

• uses Next Generation Sequencing technology on cDNA libraries generated from RNA isolated from cells.
• Generates unbiased whole transcriptome data
• Can identify novel splice variants and non-coding RNAs