BMOL20090

Polymerase chain reaction (PCR)

Outline of lecture

1. PCR
2. Oligonucleotide synthesis
3. Next generation DNA sequencing
Important fact to remember:
DNA polymerases require a primer

No primer, no enzymatic DNA synthesis.

Traditional gene cloning

1. Isolate genomic DNA

2. Construct library

3. Screen thousands or millions of recombinants for the required sequence

- A slow and laborious process.

A gene cloned in this way is amplified in vivo

i. e. inserted into a multicopy plasmid and propagated in a bacterial host.
The polymerase chain reaction rapidly amplifies
specific DNA sequences in vitro.

A DNA polymerase is used to synthesise billions of copies

of the required sequence in a tube.
Lecture by Kary Mullis on discovery of PCR:

http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html

Kary Mullis
28th December 1944 - 7th August 2019
The polymerase chain reaction (PCR)

amplifies specific sequences of DNA in vitro.

A pair of oligonucleotides is used to prime enzymatic

synthesis of both strands of the target sequence.

Target sequence
(region to be amplified)
The new duplexes are denatured and a second cycle
of synthesis takes place.

Cycle 2 gives 4 (22) copies .

Cycle 3 gives 8 (23) copies .
Cycle 4 gives
16 (24) copies .
With multiple cycles of annealing, extension and denaturation,
the number of copies of the target sequence increases exponentially.
Components of a polymerase chain reaction mixture:

Oligonucleotide primers

Template DNA

All 4 dNTPs

Heat-stable DNA polymerase

Buffer
The mixture is placed in a programmable heating block
and subjected to ~ 30 cycles of denaturation, annealing
and extension.

Nanogram amounts of DNA can be generated from a single

template molecule.
Typical thermal cycling programme

Denaturation

95°C 3 min 95°C 1 min

Extension

Initial
denaturation 72°C 1 min 72°C 10 min

Final
extension

65°C 1 min

Annealing

~ 30 cycles
Agarose gel electrophoresis of PCR products
amplified from bacterial chromosomal DNA.

PCR product
bands
Standard PCR

The final amount of amplified DNA is the same

whether the original template is abundant or scarce.

High copy no template Low copy no template

Same amount of amplified DNA

at the end point.
Quantitative or Real-time PCR

The reaction mixture includes a “reporter” compound.

This fluoresces when it binds double-stranded DNA.

Fluorescence ↑ as the amount of amplified DNA ↑

Abundant template Low copy no template

-Product appears early -Product appears late
Fluorescence

[Amount of
PCR product]

10 20 30 No of cycles

The point at which fluorescence appears

is related to the initial number of template molecules.
Various methods are available for quantifying amplified DNA using fluorescence.

The DNA polymerase also has a 5′ to 3′ exonuclease domain.

This degrades a probe annealed to the template.

Separation of fluor and quencher results in fluorescence.

Image
By User:Braindamaged - Own work by the original uploader, Public Domain,
https://commons.wikimedia.org/w/index.php?curid=42613619
Reverse transcriptase PCR

is used to amplify DNA copies of an RNA molecule.

RNA is used as the initial template.

It is copied into DNA using reverse transcriptase

Then normal PCR is carried out.
Use of RT-PCR to assess whether a gene is transcribed
in different cells or tissues

1. Purify total mRNA,

eliminate residual DNA using RNAse-free DNAse

G AAAAAA

G AAAAAA

G AAAAAA

G AAAAAA
2. Make a DNA copies of RNA strands using reverse transcriptase and oligo dT primers

G AAAAAA
TTTTTT

G AAAAAA
TTTTTT

G AAAAAA
TTTTTT

G AAAAAA
TTTTTT

3. Amplify the sequence of interest from cDNA by normal PCR using specific primers

G AAAAAA
TTTTTT
Limitations of PCR

1. It is necessary to have specific primers, or know sequences of

the ends of the target DNA

2. It can be difficult to amplify very long sequences ( > 15kb)

although “long-range” technologies are improving

3. It can be error-prone (some heat-stable DNA polymerases

don’t proof-read and misincorporate bases)

4. The high sensitivity means that DNA contamination can be

a problem (e. g. in forensics, ancient DNA analysis).

Problems 1 -3 do not arise with traditional cloning.

There are numerous applications of PCR:

molecular archaeology, clinical microbiology, forensics,

research.....................
Why did no one think
of PCR before 1985?

Har Gobind Khorana 1922 – 2011

Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG (1971).

Studies on polynucleotides. XCVI. Repair replications of short synthetic DNAs
as catalyzed by DNA polymerases. J. Mol. Biol. 56, 341-61

1971 paper from Khorana’s group mainly examines primer

requirements of E. coli DNA polymerase I and two related enzymes

The discussion section proposes that a method essentially identical to PCR

might give multiple copies of a DNA sequence in vitro.

They did not follow up the idea.

Oligonucleotide synthesis

Methods for chemical synthesis of DNA have been automated.

1. Fill in online order

form correctly.

2. E-mail order to company

that sells oligonucleotides.

3. Arrange payment
~ €1 per base.
4. Oligonucleotides arrive by post.
 Information sheet

Gel analysis

Yield (amount)
Next –generation DNA sequencing

Limitations on Sanger sequencing

1. To provide templates,
insert fragments must be cloned into E. coli plasmid vectors.

2. Each sequencing reaction

must be analysed by electrophoresis.

Some Next-Gen technologies greatly increase

the number of templates that can be sequenced simultaneously.

They allow “massively parallel” sequencing

Next –generation DNA sequencing

Illumina technology

Fragments of genomic DNA are phosphorylated on the 5ʹ ends,

and adenosine nucleotides are added to the 3ʹ ends.
5ʹ 3ʹ

P A
5ʹ 3ʹ
A P
P A
3ʹ 5ʹ
A P

3ʹ 5ʹ

5ʹ 3ʹ

P A

A P

3ʹ 5ʹ
Synthetic adaptors are ligated to fragments of genomic DNA.

Adaptors are partly single-stranded, partly double-stranded,

and have a “T” overhang on the 3ʹ ends.

5ʹ 3ʹ

3ʹ 5ʹ 3ʹ 5ʹ

T P A P

P A P T
5ʹ 3ʹ 5ʹ 3ʹ

3ʹ 5ʹ
After ligation, each strand has different primer-binding sequences at its 5ʹ and 3ʹ ends
(represented here as red and blue).

5ʹ 3ʹ

T A

A T

5ʹ
3ʹ

Duplexes are denatured and single strands are amplified.

Each single strand is annealed to a complementary adaptor sequence covalently
linked to the surface of a flow cell.

5ʹ

3ʹ

3ʹ

A DNA polymerase synthesises the complementary strand.

The new duplex is denatured and the original strand is washed away.

3ʹ

The red adaptor sequence anneals to its immobilised complement.

The DNA polymerase synthesises the complementary strand,
then the duplex is denatured.

Synthesise 2nd strand Denature

Adaptors anneal to
immobilised complementary
sequences

DNA polymerase synthesizes

more copies of both strands.
Annealing and synthesis
are repeated.

New copies form a cluster

on the flow cell surface.
The amplification process
is like PCR
The amplification process
is like PCR.

Enough copies of the

template build up
quickly.
Multiple cycles of “bridge amplification” give a cluster containing
numerous copies of a single template molecule.
 Eventually, there are enough copies of the original strand
to provide a sequencing template.

The reverse strands are chemically cleaved from the flow cell surface.
The immobilised adaptor has a cleavable bond.
The flow cell surface is covered with millions of clusters of
different amplified template sequences.

These can all be sequenced simultaneously.

A primer is annealed to each copy of the clonally amplified template.

3ʹ

DNA polymerase incorporates a reversible chain terminator.

“Reversible terminators” allow one round of chain extension.

Base Cleavable linker Base-specific dye

G C T A

Cleavable blocking group

A fluorescent base-specific dye identifies the nucleotide incorporated.

The dye is cleaved off, the 3′ OH is unblocked,

then the next reversible terminator nucleotide is incorporated.
Structure of reversible terminator analogue of dTTP.

dTTP

Fluor

"Reversible terminator" analogue of dTTP

DNA

After incorporation, the dye is cleaved off

and the 3' OH is unblocked
The nucleotide incorporated in the first cycle is identified from its colour.

The fluorescent dye and the 3′ OH blocking group are cleaved off.

A second cycle is carried out.

4 different clusters
(Clonally amplified single template species).

Incorporate nucleotide.
Identify base.
Cleave off dye.
Unblock 3′ OH

Repeat

Repeat

Repeat

Repeat
Nanopore sequencing

A DNA strand is threaded through a transmembrane protein channel.

Each of the 4 nucleobases causes a characteristic reduction in an
electrical current through the nanopore.

This allows each base in the sequence to be identified.

Image: Nik Spencer/Nature

Eisenstein M. 2017. Nature 550:285–288

doi.org/10.1038/550285a
Main points

1. PCR is an important method for enzymatic synthesis of multiple

copies of a DNA sequence in vitro.

2. PCR and many types of DNA sequencing rely on

knowledge of DNA replication.

3. Many next-generation DNA sequencing technologies:

(a) generate sequencing templates by in vitro amplification,

which is faster than cloning in bacteria.

(b) use base-calling methods that allow massive numbers of templates

to be sequenced in parallel.

4. Nanopore sequencing exploits helicases and transmembrane channel proteins

but not DNA replication.
Students please note:

Because of time constraints,

we may have to leave out pyrosequencing.

It is becoming obsolete, but it does exploit knowledge

of DNA replication and other biological phenomena
such as bioluminescence.
Pyrosequencing

Principle of pyrosequencing method

DNA fragment to be sequenced

3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGT 3’

Primer
 DNA polymerase

3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGT

dNTPs are washed through the reaction vessel in the order:

1. dCTP
2. dGTP
3. dATPS
4. dTTP
 DNA polymerase

3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGT
dTTP
dGTP
dCTP
dATPS

In the dTTP cycle,

thymidine is incorporated and pyrophosphate is released.
 3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGTT

PPi
An enzyme system couples
pyrophosphate release
The base is recorded to light emission.
as T

T
 3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGTTC

Incorporation of C
PPi

T C
 3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGTTCG

Incorporation of G
PPi

T C G
3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGTTCGA

PPi Incorporation of A

T C G A
3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGTTCGATTT

Incorporation of 3 T residues

PPi PPi PPi

T C G A T T T
3 X the number of photons
 3’ CATTTTGCTGCCGGTCAAGCTAAAGGTAGCCCAATTTCGCG 5’
5’ GTAAAACGACGGCCAGTTCGATTTCC

PPi PPi

T C G A T T T C C
The output is called a pyrogram.
Pyrogram

The sequence determined is:

5′ GCAGGCCT 3′

In this case the flow cycle is G, C, T, A, G, C, T, A, G, C, T, A ........

Apyrase removes unused dNTPs and residual ATP before the next cycle

dNTP → dNDP + Pi → dNMP + Pi

ATP → ADP + Pi → AMP + Pi

Coupling pyrophosphate production to light emission

1. Sulfurylase

PPi + adenosine phosphosulphate Sulphate + ATP

2. Luciferase

ATP + luciferin AMP + 2Pi + oxyluciferin + LIGHT

Reaction catalysed by
Pyrophosphate
sulfurylase

APS = Adenosine
phosphosulphate

APS

The DNA polymerase

doesn’t incorporate
rATP into DNA.

ATP

Sulphate
Reaction catalysed by
luciferase

Luciferin Oxyluciferin

OH
O-
N N N N

-O S S -O S S

+ ATP + AMP
+ O2 + 2Pi
+ CO2 + H2O

+ Light
The sequencing reaction uses dATPS which cannot be used
by luciferase.

Sulphur atom

O
O- NH2
S
P N
HO P
O P
-O O O N
O -O CH2 N
O
N

OH
 NH2
O-
N
P
O N
O
HO CH2 N
O
N

dATPS is incorporated P
O
S-
normally into DNA. O NH2
N

H2C N
O N
N

OH
There is no interference between the DNA synthesis reaction
and light-emitting reaction

The DNA polymerase uses dATPS (luciferase doesn’t)

Sulfurylase generates ATP

Luciferase uses ATP (DNA polymerase doesn’t).
Pyrosequencing currently only gives ~ 200 to 300 nucleotides per template.

Sanger sequencing gives up to 1000.

However, for identifying nucleotides, measuring light emission

is easier than electrophoresis.

Thousands of pyrosequencing reactions can be carried out simultaneously

in multiwell trays.
Each well contains one species of template.

Pyrosequencing reagents are washed over the plate in each cycle.

Light pulses are captured by optical fibres beneath each well.

Large amounts of sequence data

are generated because thousands of templates are sequenced in parallel.
Ion torrent or semi-conductor sequencing
is similar to pyrosequencing, except
that nucleotide incorporation is detected by release of H+ rather than pyrophosphate.

Guanine

Guanine
H
H+

Adenine
Adenine

A proton-sensitive semiconductor is used to detect H+ release.