Unit 5: Methods and techniques

DNA microarray, qPCR, RFLP, RAPD and AFLP techniques,

FISH, chromosome painting, karyotyping, luciferase assay,
mutagenesis and deletion techniques for identification of regulatory
regions, Ames test.
 A Review of Probability

A COIN THROW
The probability of a heads (H) or a tails (T) is always 0.5 for every
throw. What is the probability of getting this combination of tails in a
row?

Event Probability
Tails
T,T
T,T,T
T,T,T,T,T
T,T,T,T,T,T,T,T,T,T,T
T,T,T,T,T,T,T,T,T,T,T,T,T,T,T,T

.
 Polymerase Chain Reaction (PCR)

• PCR is a means to amplify a particular piece of DNA

• Amplify= making numerous copies of a
segment of DNA
• PCR can make billions of copies of a target sequence of
DNA in a few hours

• PCR was invented in the 1983 Kary Mullis as a way to

make numerous copies of DNA fragments in the
laboratory

• Its applications are vast and PCR is now an integral part

of Molecular Biology
 DNA Replication vs. PCR
• PCR is a laboratory version of DNA Replication in cells
• The laboratory version is commonly called “in vitro”
since it occurs in a test tube while “in vivo” signifies
occurring in a living cell.

Key enzymes involved in DNA Replication

• DNA Polymerase (Taq Polymerase)

• DNA Ligase
• Primase
• Helicase
• Topoisomerase
• Single strand binding protein
 PCR: the in vitro version of DNA Replication

The following components are needed to perform

PCR in the laboratory:
1) DNA (your DNA of interest that contains the target
sequence you wish to copy)
2) A heat-stable DNA Polymerase (like Taq Polymerase)
3) All four nucleotide triphosphates
4) Buffers
5) Two short, single-stranded DNA molecules that serve as
primers
6) Thin walled tubes
7) Thermal cycler (a device that can change temperatures
dramatically in a very short period of time)
 PCR

The DNA, DNA

polymerase, buffer,
nucleoside triphosphates,
and primers are placed in
a thin-walled tube and
then these tubes are
placed in the PCR
thermal cycler

PCR Thermocycler
 Reaction components for PCR

1. Taq Polymerase

2. Template DNA

3. Primers (Forward and Reverse primer)

4. DNTPs

5. MgCl2

6. Buffer (Taq buffer)

7. H20

8. SYBR green or Taqman probes

 The three main steps of PCR
• The basis of PCR is temperature changes and the effect that these
temperature changes have on the DNA.

• In a PCR reaction, the following series of steps is repeated 20-40 times

(note: 25 cycles usually takes about 2 hours and amplifies the DNA
fragment of interest 100,000 fold)

Step 1: Denature DNA

At 95°C, the DNA is denatured (i.e. the two strands are separated)

Step 2: Primers Anneal

At 40°C- 65°C, the primers anneal (or bind to) their complementary
sequences on the single strands of DNA

Step 3: DNA polymerase Extends the DNA chain

At 72°C, DNA Polymerase extends the DNA chain by adding nucleotides to
the 3’ ends of the primers.
 Heat-stable DNA Polymerase

• Given that PCR involves very high temperatures, it is

imperative that a heat-stable DNA polymerase be used
in the reaction.
• Most DNA polymerases would denature (and thus
not function properly) at the high temperatures of
PCR.
• Taq DNA polymerase was purified from the hot springs
bacterium Thermus aquaticus in 1976
• Taq has maximal enzymatic activity at 75 °C to 80 °C,
and substantially reduced activities at lower
temperatures.
 PCR Conditions

Step-1

Step-3 Step-3
Step 1 Denaturation of DNA

This occurs at 95 ºC mimicking the function of

helicase in the cell.
Step 2 Annealing or Primers Binding

Reverse Primer

Forward Primer

Primers bind to the complimentary sequence on the

target DNA. Primers are chosen such that one is
complimentary to the one strand at one end of the
target sequence and that the other is complimentary
to the other strand at the other end of the target
sequence.
Step 3 Extension or Primer Extension

extension

extension

DNA polymerase catalyzes the extension of the

strand in the 5-3 direction, starting at the
primers, attaching the appropriate nucleotide
(A-T, C-G)
• The next cycle will begin by denaturing
the new DNA strands formed in the
previous cycle

Formula: 2n, where n is the number of cycles

 Tutorial

https://dnalc.cshl.edu/resources/animations/pcr.html
 Exercise

Initial denaturation: 5 min

Denaturation: 30 sec

Aneeling: 30 sec 30 cycles

Extension 60 seconds

Final annealing: 5 min

30 x 30= 900 sec or 15 mins

60 x 30 = 1800 sec or 30 minutes

Total time taken to complete 30 cycle PCR reaction: 5+15+15+30+5 = 70 min

The DNA of interest is amplified by a power of 2 for each PCR
cycle

For example, if you subject your DNA of interest to 5 cycles of

PCR, you will end up with 25 (or 64) copies of DNA.

If you subject your DNA of interest to 35 cycles of PCR, how

many copies of DNA will you obtain?
 More about Primers
• PCR primers are short, single stranded DNA molecules
(18-25 bp).

• They are manufactured commercially and can be ordered

to match any DNA sequence.

• Primers are sequence specific, they will bind to a particular

sequence in a genome.

• As you design primers with a longer length (18 → 25 bp),

the primers become more selective.

• DNA polymerase requires primers to initiate replication

 Selectivity of Primers
• Primers bind to their complementary sequence on the target DNA

– A primer composed of only 3 letter, ACC, for example, would be

very likely to encounter its complement in a genome.

– As the size of the primer is increased, the likelihood of, for

example, a primer sequence of 20 base letters repeatedly
encountering a perfect complementary section on the target DNA
become remote.
 Probability in Genetics
• There are 4 bases in the DNA molecule A,C,G,T
• The probability of encountering any of these bases in the code is 0.25 (1/4)
• So let us look at the probability of encountering a particular sequence of bases

Event Probability
A 0.25 = 0.25
A,T 0.25 x 0.25 = 0.0625
A,T,A 0.25 x0.25 x 0.25 = 0.015625
A,T,A,G,G (0.25)5 = 0.0009765
11
A,T,A,G,G,T,T,T,A,A,C (0.25) = 0.000002384
16
A,T,A,G,G,T,T,T,A,A,C,C,T,G,G,T (0.25) =0.0000000002384

So it become increasing unlikely that one will get 16 bases in this particular
sequence (1 chance in 4.3 billion). In this same way, one can see that as the
primer increases in size, the chances of a match other than the one intended
for is highly unlikely.
 PCR links

• The Dolan DNA Learning Center:

Link to Dolan DNA Learning Center's PCR Animation
This site provides a nice step by step guide to how DNA is copied in
PCR reaction.
• DNA Interactive:
Link to DNAi
This site is FULL of cool stuff! Two 3-D animation videos relevant to
this Powerpoint presentation are:
– The DNA Replication animation (to get to this, click on the above
link, then click on “code”, then click on “copying the code”, then click on
“putting it together”, and finally click on “replication”).
– The PCR animation (to get to this, click on the above link, then click
on “manipulation”, then click on “techniques”, the click on “amplifying”
and then finally click on “PCR animation”).
 PCR Applications

• Primers can be created that will only bind and amplify

certain alleles of genes or mutations of genes
• This is the basis of genetic counseling and PCR is used as
part of the diagnostic tests for genetic diseases.
• Some diseases that can be diagnosed with the help of
PCR:
• COVID-19
• Huntington's disease
• cystic fibrosis
• Human immunodeficiency virus
 Huntington’s Disease (HD)
• HD is a genetic disorder characterized by abnormal body
movements and reduced mental abilities
• HD is caused by a mutation in the Huntingtin (HD) gene
• In individuals with HD, the HD gene is “expanded”
– In non-HD individuals, the HD gene has a pattern called trinucleotide
repeats with “CAG” occurring in repetition less than 30 times.
– IN HD individuals, the “CAG” trinucleotide repeat occurs more that 36
times in the HD gene
• PCR can be performed on an individual’s DNA to determine whether
the individual has HD.
– The DNA is amplified via PCR and sequenced (a technique by which
the exact nucleotide sequence is determined) and the number of
trinucleotide repeats is then counted.
 Human Immunodeficiency Virus (HIV)

• HIV is a retrovirus that attacks the immune system.

• HIV tests rely on PCR with primers that will only amplify
a section of the viral DNA found in an infected
individual’s bodily fluids.
Therefore if there is a PCR product, the person is likely to be
HIV positive. If there is no PCR product the person is likely to
be HIV negative.
• Protein detection based tests are available as well but all US blood
is tested by PCR.
 PCR and Forensic Science
• Forensic science is the application of a broad spectrum of sciences
to answer questions of interest to the legal system. This may be in
relation to a crime or to a civil action.

• It is often of interest in forensic science to identify individuals

genetically. In these cases, one is interested in looking at variable
regions of the genome as opposed to highly-conserved genes.

• PCR can be used to amplify highly variable regions of the human

genome. These regions contain runs of short, repeated sequences
(known as variable number of tandem repeat (VNTR) sequences) .
The number of repeats can vary from 4-40 in different individuals.

• Primers are chosen that will amplify these repeated areas and the
genomic fragments generated give us a unique “genetic fingerprint”
that can be used to identify an individual.
PCR Applications to Forensic Science

• Paternity testing
• DNA finger printing

• Identifying badly decomposed bodies or when only

body fragments are found - World trade center,
Bosnian , Iraq & Rwandan mass graves