PCR, Theory and Applications
Davidson Day Ateh
Neuroscience Centre, Institute of Cell and Molecular Sciences
Barts and The London School of Medicine and Dentistry
Queen Mary University of London
Experimental Neuropathology Module - November 2007
Intercalated Experimental Pathology BSc
DNA
DNA Replication In Vivo
DNA replication occurs during cell division
• Different types of DNA polymerase (e.g.
I, II & III and those involved in DNA repair)
• RNA polymerase for transcription
• High fidelity DNA synthesis is due to proof
reading (only one error per 1  109 nucleotides)
DNA Polymerase facilitates replication
Polymerase Chain Reaction
Or in vitro DNA replication
• Gene cloning (recombinant DNA
techniques) developed in the 1970’s
• PCR originally a slow, labour intensive
process that required the addition of fresh
DNA Polymerase every replication round
Thermus Aquaticus was discovered in the
Yellowstone (USA) hotsprings in the 1960’s
and thrives at around 72°C
• Revolutionised by Kary Mullis in the 1980’s whilst
he was working for a biotechnology company
• He received the 1993 Nobel Prize in
Chemistry for his work
• A DNA polymerase (Taq) is used to
make many copies of a short length of
DNA defined by primers in a test tube
• Taq DNA polymerase
works optimally at 72°C
• Critically it is not
denatured at 94°C
(thermostable)
ONE OF THE MOST IMPORTANT MOLECULAR BIOLOGY TECHNIQUES
PCR Mix
• Template DNA
• Buffer (with Mg2+)
• Nucleotides (dNTPs)
• Taq DNA Polymerase (or other thermostable DNA polymerase)
• Primers
PCR Mechanisms
Double Stranded
DNA
Denaturing
DNA template
Single Stranded
DNA
Complimentary
binding forward and a
reverse primers (20-30
oligonucleotides)
PCR
100
Melting
94 oC
Temperature
30x
Melting
94 oC
Extension
Annealing
Primers
50
72 oC
50 oC
0
Time
3’
5’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
5’
3’
5’
5’
3’
5’
5’
5’
5’
5’
3’
3’
5’
3’
PCR
100
Melting
94 oC
Temperature
30x
Melting
94 oC
Extension
Annealing
Primers
50
72 oC
50 oC
0
Time
3’
5’
5’
3’
3’
5’
3’
5’
5’
3’
5’
5’
5’
3’
5’
5’
3’
5’
5’
5’
5’
5’
3’
3’
5’
3’
PCR
Strands that are too long double in size
whereas strands that are just right
increase exponentially
5’
Fragments of
defined length
5’
5’
5’
5’
5’
5’
5’
DNA Amplification
Number
1
2
0
1
Cycles
4
8
16
32
64
2
3
4
5
6
PCR Yield
Theoretically, the number of DNA fragment copies obtained can be calculated
Yield = 2n  y
Where y is the initial number of DNA copies and n is the number of thermal cycles
If you start with 1000 copies, how many copies are made in 32 cycles?
2n x y
= 232 x 1000
= 4,294,967,296 x 1000
= 4,294,967,296,000
Thermal Cyclers
Challenges
Fidelity of the Reaction
• Taq DNA polymerase lacks the proof-reading activity present in other polymerases
• Taq makes 1 error per 1  104 nucleotides (remember, 1 per 1  109 nucleotides in vivo)
• Thus, a 400 base pair target will contain an error in 33% of molecules after 20 cycles
• Error distribution will be random
• Does not matter if PCR product is for sequencing or to be cut with restriction enzymes
• Does matter if you want to clone the product (use proof-reading thermostable enzyme)
Optimising the PCR Reaction
• The amount of template and polymerase
• Annealing temperature of the primers and their design
• The concentration of Mg2+ in the reaction
• The extension time and temperature
• The denaturing and annealing times
The use of PCR
• PCR is a DNA ‘amplification’ method, many copies of any DNA
template can synthesised
• One starting DNA template can be amplified in to an infinite
number of copies
• “Amplified” fragments of DNA can be sequenced, cloned, probed or
sized using electrophoresis
• Defective genes can be amplified to diagnose illnesses
• Genes from pathogens can be amplified to identify them (e.g. HIV)
• Amplified fragments can act as genetic fingerprints using restriction
enzymes (nucleases cut, shorten or degrade DNA, Ligases join
DNA, polymerases make DNA copies)
PCR Practical Example
+/+
Genotyping Loa mice
Loa/+
T-to-A transversion in the Dnchc1 gene that results in residue 580
changing from phenylalanine (TTC) to tyrosine (TAC)
PCR Practical Example
Genotyping Loa mice
DNA prep from mouse tail Biopsies
•
Cut 0.8-1.0 cm of mice tail (or equivalent mass of other parts), divide into small pieces and
transfer into Eppendorf tube
•
Add 300l of Lysis buffer and 3l of proteinase K. Incubate tubes at 55C overnight (lysis
buffer- 100mM Tris-HCl pH8.0, 5mM EDTA, 0.2% SDS, 200mM NaCl, Proteinase K stock
is 20mg/ml New England Biolabs) and leave to digest overnight
•
Vortex each tube well. Spin 10-15 min to pellet hair etc…
•
Pour supernatant into empty Eppendorf tube
•
Dilute 4l in 200l H2O and use 2l of this in 20l PCR reactions for genotyping
PCR Practical Example
PCR Mix per tube
HotStar Taq master mix (Qiagen)
MDN-Int7-F (10 uM)
MDN2064-R (10 uM)
H2O
10ul
2 ul
2 ul
4 ul
----18 ul
+ 2ul of 1:50 diluted DNA template
Genotyping Loa mice
Thermocycler
1) 95C 15min
2) 95C 30S
3) 62C 30S
4) 72C 1min
5) Go to 2, 35 times
6) 72C 10min
7) 16C Hold
PCR Practical Example
Genotyping Loa mice
T-to-A change
Forward Primer
TGCTGCTGAGCTGCGTCCTAGTGCTGTGTGCTCTCCTGTTTTCATTCCCTCTTCACAT
TCATTAGTTCTTTCCTTTAAGTATACACACACACACACACACACACACAGTAAAGACA
GAAGTCTGCAGGGAGATCCTTATAGTGTGCTCATGGCTGAATTGTGATGATAGAGTCC
TAAAGGCCTAGAAGTCAGCATTGATGCAAGAATCCTGTGCTGTGCCTGTGACAGAAAA
ACGTCATTTGCAGCTATGTTTTGTTCCAAACCTTTTGTTTTAGGTCACAGCAGTCGCA
CAACAGAACCAAGGAGAAGCACCTGAACCCCAAGACATGAAAGTGGCCGAGGTGCTCT
TTGATGCTGCCGACGCCAACGCCATTGAGGAGGTGAACCTGGCCTACGAGAATGTCAA
GGAAGTCGATGGTCTGGATGTTTCCAAAGAAGGGACGGAAGCCTGGGAGGCCGCGATG
AAGAGATACGATGAGAGGATCGACCGTGTGGAGACCCGCATCACCGCCCGCCTCCGAG
ATCAGCTCGGCACGGCCAAGAATGCCAATGAGATGTTCAGGATTTTCTCCAGGTTCAA
TGCACTGTTCGTCCGCCCACACATCCGAGGGGCCATTCGTGAATACCAGACCCAGCTG
ATCCAACGTGTGAAAGATGACATCGAATCTCTGCACGACAAGTTCAAGGTCCAGTACC
CGCAAAGCCAAGCTTGTAAAATGA
Reverse Primer
Amplified fragment is 696 bp long
PCR Practical Example
Genotyping Loa mice
PCR Product
Use agarose gels (typically 2% w/v)
Incorporate ethidium bromide or other DNA dye
PCR Product
PCR Practical Example
Genotyping Loa mice
PCR Product Digestion
Digestion with RsaI (GT|AC) at 37°C for 2 hrs
537bp
135bp
24bp
672bp
Wt
+
Loa/Loa
+/Loa
+
+
+
+
+
+
+
+
PCR Practical Example
PCR Product Digestion
wt
672 bp
537 bp
135 bp
24 bp
Loa/wt
Loa/Loa
Genotyping Loa mice
Further PCR
Examples
• Quantitative (Real-Time) PCR (Q-PCR)
• Reverse Transcription PCR (RT-PCR)
• Multiplex-PCR
• Helicase Dependant Amplification (HAD)
END