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Polymerase Chain Reaction

(PCR)

Nandana N Kumara
Wayamba University of Sri Lanka
DNA
DNA is a nucleic acid that is composed of two
complementary nucleotide building block
chains.

The nucleotides are made up of a phosphate


group, a five carbon sugar, and a nitrogen
base.
DNA
DNA has four nitrogen bases.

• Two are purines ( 2 ringed base )


– Adenine ( A ), Guanine ( G )

• Two are pyrimidines ( 1 ringed base )


– Cytosine ( C ), Thymine ( T )
DNA
These four bases are linked in a repeated
pattern by hydrogen bonding between the
nitrogen bases.

The linking of the two complementary


strands is called hybridization.
DNA
A purine always links with a pyrimidine base
to maintain the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with two


hydrogen bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with three


hydrogen bonds between them.
DNA
Example of bonding pattern.
• Primary strand

CCGAATGGGATGC
GGCTTACCCTACG

• Complementary strand
DNA Molecule
Adeni ne

Thymi ne

Guani ne

Cy tosi ne
What is the Polymerase
Chain Reaction?
• It’s a means of selectively amplifying a
particular segment of DNA.
• The segment may represent a small part of a
large and complex mixture of DNAs:
e.g. a specific exon of a human gene.
• It can be thought of as a molecular
photocopier.
How Powerful is PCR?
• PCR can amplify a usable amount of DNA
(visible by gel electrophoresis) in ~2 hours.
• The template DNA need not be highly
purified — ex: a boiled bacterial colony.
• The PCR product can be digested with
restriction enzymes, sequenced or cloned.
• PCR can amplify a single DNA molecule, e.g.
from a single sperm.
Gene Analysis Prior to PCR?
• Southern blotting (1975) permitted
rudimentary mapping of genes in unrelated
individuals (RFLPs, insertions & deletions).
• DNA sequencing (1978) required genes to
first be cloned into plasmid or λ vectors.
• Gene library construction and screening
could take many months and libraries had
to be made for each individual analyzed.
The Invention of PCR
• Invented by Kary Mullis in 1983.
• First published account appeared in 1985.
• Awarded Nobel Prize for Chemistry in 1993.
Did He Really Invent PCR?
• The basic principle of replicating a piece of
DNA using two primers had already been
described by Gobind Khorana in 1971: –
Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.
• Progress was limited by primer synthesis
and polymerase purification issues.
• Mullis properly exploited amplification.
The Basics of PCR Cycling
Each cycle comprise:
denaturation (95°C), - 30 sec.
annealing (55–60°C), - 30 sec.
extension (72°C), - time depends on product
size.
What’s in the Reaction mixture?
• Template DNA
• Reaction buffer (Tris, ammonium ions
(and/or potassium ions), magnesium ions,
bovine serum albumin)
• Nucleotides (dNTPs)
• Primers
• DNA polymerase (usually Taq)
PCR Primers
Primers range from 15 to 30 nucleotides, are
single-stranded, and are used for the
complementary building blocks of the target
sequence.
PCR Primers
• A primer for each target sequence on the
end of your DNA is needed.
• This allows both strands to be copied
simultaneously in both directions.
• DNA is copied from 3’ end to the 5’ end
PCR Primers

3’TTAACGGCCTTAA . . . TTTAAACCGGTT5’
AATTGCCGGAATT . . . . . . . . . .>
and
<. . . . . . . . . . AAATTTGGCCAA
5’TTAACGGCCTTAA . . . TTTAAACCGGTT3’
PCR Primers
The primers are added in excess so they will
bind to the target DNA instead of the two
strands binding back to each other.
PCR In Detail
• Denature, anneal, extend and repeat the
• cycle 30 to 35 times.
• How does the polymerase know to stop?
“when it reaches the other primer”

Most textbooks do not fully explain PCR.


• PCR animation at Dolan DNA Learning Center,
How many copies?
• No target products are made until the third
cycle.
• The accumulation is not strictly a doubling
at each cycle in the early phase.
• If there were 2 DNA molecules at the
beginning, at 30 cycles there will be 230
copies. (1073741824)
How many cycles?
• Increasing the cycle number above ~35
has little positive effect.
• The plateau occurs when:
The reagents are depleted
The products re-anneal
The polymerase is damaged
• Unwanted products accumulate.
So Then, it’s Easy?
• Cycling performed with three water baths.
• Thermal cyclers introduced in 1986.
• Early polymerases were not thermostable,
so had to be replenished each cycle.
• The 37°C temperature caused non-specific
priming, resulting in unwanted products.
• Taq (Thermus aquaticus) DNA polymerase
first described in 1988.
Thermal Cyclers
• PCR cyclers available from many suppliers.
• Many block formats and multi-block
systems.
• Reactions in tubes or 96-well micro-titre
plates.
So, I Can Just Go Ahead?
• Not so fast.
• The PCR technique and the use of Taq DNA
polymerase in PCR are both patented.
• Even academic and public organisations
must pay license fees
• levy paid on enzyme and thermal cycler
purchases.
Has It Worked?
• Check a sample by gel electrophoresis.
• Is the product the size that you expected?
• Is there more than one band?
• Is any band the correct size?
• May need to optimize the reaction
conditions.
Optimising the PCR Reaction
• Annealing temperature of the primers
• The concentration of Mg2+ in the reaction
• The extension time
• The denaturing and annealing times
• The extension temperature
• The amount of template and polymerase
Optimising the Annealing
Temperature
• Primers have a calculated annealing
temperature (e.g. 54°C)
• Temperature must be confirmed
practically.
• Temperature steps of 2°C above and below
• Use gradient cycler
Optimising the Mg2+
Concentration
• The fidelity of the PCR depends on [Mg2+].
• Vary [Mg2+] in steps of 0.5 mMol.
• Sometimes a compromise between yield
and specificity.
Fidelity of the Reaction
• Taq DNA polymerase lacks the 3´→5´
proof-reading activity commonly present in
other polymerases.
• Taq mis-incorporates 1 base in 104.
• A 400 bp target will contain an error in 33%
of molecules after 20 cycles.
• Error distribution will be random.
Do Errors Matter?
• Yes, if you want to clone the amplified DNA
— an individual molecule may harbour
several mutations.
• No, if you want to sequence the amplified
DNA or cut it with restriction enzymes.
• Use a proof-reading thermo-stable enzyme
rather than Taq.
How Big A Target?
• Amplification products are typically in the
size range 100-1500 bp.
• Longer targets are amplifiable — >25 kb.
• Requires modified reaction buffer, ocktails
of polymerases, and longer extension
times.
Can I PCR Amplify RNA?
• Not directly — the DNA polymerase
requires a DNA template and will not copy
RNA.
• mRNA can first be copied into cDNA using
reverse transcriptase.
• cDNA is a template for PCR —it need not be
double-stranded.
Cloning PCR Products
• Products should be ligatable into
bluntended restriction enzyme site.
• Lower than expected efficiency.
• Products are not truly blunt-ended.
• Taq polymerase adds a single non-
templated base (usually A) to the 3´ end:
NNNNNNN…NNNNNNNA
ANNNNNNN…NNNNNNN
Designing PCR Primers
• Primers should be ~20 bases long.
• The G/C content should be 45–55%.
• The annealing temperatures should be
within 1°C of one another.
• The 3´-most base should be a G or C.
• The primers must not base pair with each
other or with themselves or form hairpins.
• Primers must avoid repetitive DNA regions.
Taq DNA polymerase
• Extracted from Thermus aquaticus
• Thermo-stable polymerases
• Not permanently destroyed at 94ºC
• Optimal temperature is 72ºC
Problems with Taq
• Does not have proof reading ability
• Error rate 1 in 2 X 104 bases
• Seems rare but can be recovered in cloning
a single molecule
• Newer polymerases have high fidelity
Thermo-stable DNA polymerases