Introduction to
Polymerase Chain
Reaction (PCR)
Review: The structure of
DNA
Unzipping
Antiparallel Strands
Review: Genome Sizes
Pine: 68 billion bp
Corn: 5.0 billion bp
Soybean: 1.1 billion bp
Human: 3.4 billion bp
Housefly: 900 million
bp
Rice: 400 million bp
E. coli: 4.6 million bp
HIV: 9.7 thousand bp
http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt
The Problem...
How do we identify and detect
a specific sequence in a
genome?
The Problem...
(How do we identify and detect a
specific sequence in a genome?)
TWO BIG ISSUES:
There are a LOT of other sequences
)
I Y
T
IFI
C in a genome that we’re not
P EC interested in detecting.
(S
The amount of DNA in samples
N )
T O
Iwe’re interested in is VERY small.
A
IC
L I F
M P
(A
Just How Big Is 3.4 Billion?
Human genome is 3.4 billion
bp
If the bases were written in
standard 10-point type, on a
tape measure...
...The tape would stretch for
5,366 MILES = 8,636 km!
Identifying a 500bp
sequence in a genome would
be like finding a section of
this tape measure only 4 feet
long = 122 cm!
How many molecules do we
need to be able to see them?
To be visible on an agarose gel, need
around 10 ng DNA
For a 500-bp product band, weighing
660 g/mol.bp, therefore need 10 x 10-
9
/ (500*660) = 3.03x 10-14 moles
Avogadro’s number = 6.02 x 1023
Therefore need 1.8 x 1010 copies!
In other words, to “see” a single
“gene”, the DNA in a sample of
100 cells would have to be
multiplied 180 million times!!!!!
The Problem...
(How do we identify and detect a
specific sequence in a genome?)
TWO BIG ISSUES:
There are a LOT of other sequences
I Tin
Y a genome that we’re not
F I C
E CI interested in detecting.
SP The amount of DNA in samples
I O N
A Twe’re interested in is VERY small.
F I C
P LI
AM
PCR solves BOTH of these issues!!!
What is PCR?
PCR (Polymerase Chain Reaction)
is a process used to make many
copies of select regions of DNA.
Inventor: 1983 Kary Mullis
Nobel prize in chemistry in 1993
Some Uses of PCR
Forensic DNA detection
Proving innocence
Identifying transgenic plants
Detection of diseases
Cloning
Detection of ancient DNA
Specific sequence targeting
Assembling artificial sequences
Endangered spsecies
GMO detection
The Process of PCR
Consists of three steps:
Denaturation
Annealing
Extension
Denaturation
The double-stranded DNA has to be
heated to 94-96°C in order to
separate the strands
The more there is G or C, the higher
Tm
The longer the primers, the higer Tm
Annealing
The temperature is lowered so the
primers can attach themselves to
the single DNA strands.
The temperature depends on:
Concentration of primers
Composition of nucleotides
Building starts from the 3’ end
Extension
The DNA-Polymerase has to copy
the DNA strands.
It starts at the annealed primer
and works its way along the DNA
strand.
The extension temperature
depends on the DNA-Polymerase.
What time does it take?
Denaturation: 30 - 60 sec
Annealing: 30 - 60 sec
Extension: 30 - 60 sec
25 - 35 cycles only (otherwise enzyme
decay causes artifacts)
72oC for 5 min at end to allow complete
elongation of all product DNA
Altogether: 7 min ( 8,5 min) * 25 (35) = 3h-
5h
How PCR works
The PCR cycle
Denaturation 93°-95°C
Annealing usually at 50° to 70°C
depending on the Tm of the oligos
Extension: DNA synthesis which is
about 70 - 75°C.
How PCR works
How PCR works
How PCR works
PCR Reaction Components
Water
Buffer solution
DNA template
Primers
Nucleotides
Mg++ ions
DNA
Polymerase
PCR Reaction: Water
Water
The medium for all other
components.
PCR Reaction: Buffer
Buffer
Stabilizes the DNA polymerase,
DNA, and nucleotides
500 mM KCl
100 mM Tris-HCl, pH 8.3
Triton X-100 or Tween
PCR Reaction: Template DNA
DNA template
Contains region to
be amplified
Any DNA desired
Purity not
required
Should be free of
polymerase
inhibitors
PCR Reaction: Primers
Primers
Specific for ends of
amplified region
Forward and Reverse
Annealing temps
should be known
Depends on primer length,
GC content, etc.
Length 15-30 nt
Conc 0.1 – 1.0 µM (pMol/µL)
PCR Reaction: Nucleotides
Nucleotides
Added to the
growing chain
Activated NTP’s
dATP, dGTP, dCTP, dTTP
Stored at 10 mM, pH 7.0
Add to 20-200 µM in
assay
PCR Reaction: Magnesium
Mg++ ions
Essential
co-factor of DNA polymerase
Too little: Enzyme won’t
work.
Stabilizes the DNA double-
helix
Too much: DNA extra
stable, non-specific priming,
band smearing
Used at 0.5 to 3.5 µM in the
assay
PCR Reaction: Polymerase
DNA
Polymerase
The enzyme
that does the
extension
TAQ or similar
Heat-stable
Approx 1 U /
rxn
A Typical PCR Reaction
Sterile Water 38.0 µL
10X PCR Buffer 5.0 µL
MgCl2 (50mM) 2.5 µL
dNTP’s (10mM each) 1.0 µL
PrimerFWD (25 pmol/µL) 1.0 µL
PrimerREV 1.0 µL
DNA Polymerase 0.5 µL
DNA Template 1.0 µL
Total Volume 50.0 µL
A Simpler PCR Reaction
Sterile Water 38.0 µL
10X PCR Buffer 5.0 µL
MgCl2 (50mM) 2.5 µL
dNTP’s (10mM each) 1.0 µL
PrimerFWD (25 pmol/µL) 1.0 µL
PrimerREV 1.0 µL
PREMIX 24.0 µL
DNA Polymerase 0.5 µL
DNA Template 1.0 µL
Buffer
MgCl2
Total Volume 50.0 µL dNTP’s
DNA Polymerase
“Enhancers”
PREMIXES CAN Sterile Water
Primers FWD+Rev 1.0 µL
REDUCE THE DNA Template 25.0 µL
NUMBER OF
ITEMS ADDED Total Volume 50.0 µL
TO THE MIX
Using a PCR Mastermix
Component 1X 20X
Sterile Water 38.0 µL 760 ul
10X PCR Buffer 5.0 µL 100 µL
MgCl2 (50mM) 2.5 µL 50 µL
dNTP’s (10mM each) 1.0 µL 20 µL
PrimerFWD (25 pmol/ul) 1.0 µL 20 µL
PrimerREV 1.0 µL 20 µL
DNA Polymerase 0.5 µL 10 µL
DNA Template 1.0 µL --
Aliquot
Total Volume 50.0 µL 980 µL 49 µL
Add DNA
as last step
Typical Thermal Cycler
Conditions
1. Initial Denaturation 95o C 4 min
2. DNA Denaturation 95o C 1 min
3. Primer Annealing 65o C 1 min
4. Primer Extension 72o C 1 min
5. Go to step #2, repeat 29 more times
6. Hold at 4o C
7. End
Thermal Cycler/PCR Machine
End of PCR Introduction
