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