Contents
1. Hospital
11. How much DNA?
2. PCR gel
12. How many cycles?
3. Results
13. Apparatus
4. Eppendorf
14. Taq, PFU
5. EB gel
15. Glossary
6. K Mullis
16. Cycling parameters
7. Big issue
17. 3’ end for specificity
8. PCR 1-2
18. Bad primers
9. PCR 3
19. Hot start
10. PCR Summary
20. Nested primers
21. Alder
Slide 1
Interpretation of DNA tests
There was a fire in the maternity Hospital and the mothers and new
babies had to be evacuated. After the fire was out and all the mums
and babies were safely back in the wards, one mother insisted that the
baby boy she had been given was NOT hers.
DNA was isolated from samples taken from this mother and
the father of her baby, and also from the five boys in the ward. A
very small fragment of the DNA was then amplified (copied) using PCR
(Polymerase Chain Reaction). The fragment is used for forensic tests
because its size is variable and because it is present in two copies,
one from each parent, in all humans. The variability is caused by
internal duplication of a 16 bp (base pair) sequence of DNA. The 16
bp sequence is repeated between 14 and 41 times in different peoples
genomes. (All but the 15-mer repeats occur frequently.)
Can you discover which of the boys belongs to the parents using
the test results shown in the figure blow ?
Slide 2
large DNA
fragments
Father
Allelic
ladder
Mother
Agarose gel for separating DNA fragments according to size
Allelic
ladder 1
Boys
2
3
4
Migration of DNA
+
small DNA
fragments
Which boy belongs to the mother and father?
Slide 3
Allelic
5 ladder
Slide 4
Father
Mother
Allelic
ladder
Allelic
ladder 1
Boys
2
3
4
Allelic
5 ladder
PCR
Polymerase Chain Reaction
Method for amplifying DNA fragments
• In vitro alternative to DNA cloning
Slide 5
Kary B Mullis
Day 0
Investment
Day 1
Profit
Double investment every day
Slide 6
10 days
210 = £ 1024
20 days
220 = £ 1 million (106)
30 days
230 = £ 1 billion
40 days
240 = 1 trillion 1012
(103)
(109)
PCR reactions 1 and 2
3’
Primer 1
5’
DNA polymerase
dNTPs
ss DNA,
“Template”
known sequence
95 C
(to denature DNA)
Primers 1+2
DNA polymerase
dNTPs
known sequence
Primer 2
Slide 7
2 partially
doublestranded
DNA
molecules
PCR reactions 3
95 C
Primers 1+2
DNA polymerase
dNTPs
PCR fragment
dsDNA, specific lenght
partially ds DNA
Slide 8
Summary PCR reactions 1-3
dsDNA
Template
Primers 1+2
dNTPs
3x 95 C denaturation
3x DNA polymerase
2 dsDNA PCR fragments
of specific length
From now on...
Every denaturation-polymerisation cycle (i) doubles the
number (n) of PCR fragments
i-3
i-2(+2
nn==22i-2
(+2 i-3))
…………. until dNTPs or primers run out, or the enzyme
activity becomes limiting.
Slide 9
How much DNA can be synthesised ?
100 µl PCR reaction
Ingredient pmoles
Molecules
Capacity
dNTPs
800
5•1014
0.4 µg dsDNA
Primers
2 x 50
2 x 3•1013
33 µg 1 kb DNA
dNTPs are limiting
0.4 µg dsDNA 1 kb fragment = 0.7 pmoles = 4x1011 molecules
- sufficient for 10 strong bands on agarose gel
- sufficient for several cloning experiments
How many PCR cycles starting from a single template molecule ?
40+2 cycles (c. 4 h in PCR machine)
Slide 10
How many PCR cycles are required to synthesise 1
g DNA from 1 ng of template DNA?
Running too many PCR cycles creates artefacts!
1 pmole = 6•1011 molecules
Slide 11
PCR
Applications of PCR
• DNA fingerprinting
• RFLP mapping
• Detection and identification of pathogenic bacteria
• DNA for sequencing and cloning
• Joining sequence contigs “gap filling”
Contig 1
Slide 12
Gap <10 kb
Contig 2
Heat-stable DNA polymerases for PCR
Elongation at 72 °C
Survive 95 °C
100 x slower than PolIII. Allow c. 1 min/kb for PCR
Taq (Thermus aquaticus) polymerase:
no proofreading
Yellowstone
Deep sea vent
Pfu (Pyrococcus furiosus) has proofreading 3’>5’
exonuclease, fewer bp changes than Taq but primer
shortening reduces specificity. Pfu is more heat
stable and more processive than Taq
All DNA polymerases require optimal free [Mg2+]
DNA and dNTPs bind Mg2+!
Slide 13
1
dsDNA
two strands, opposite polarity
2
denaturation
separating the DNA strands
3
annealing
complementary DNA strands join together to form
perfectly matched double-stranded DNA
4
Tm (primer)
melting temperature of primer-template complex
5
annealing
temperature
lowest temperature during PCR cycle
6
DNA primer
ca 20 nucleotide single-stranded DNA (synthetic
oligo-nucleotide)
7
priming
providing partially double-stranded DNA as
substrate for DNA polymerase
8
elongation
synthesising a complementary DNA strand
9
PCR cycle
95° , 55°, 72° 30 sec to 2 min each
Slide 14
PCR primer specificity
PCR can be used to amplify a specific fragments from total
genomic DNA if the priming sites are unique, and the annealing
conditions are optimal
Annealing temperature of primers: Tm (ºC)  4 x (G+C) +2 x (A+T)
(there are more complicated formulae but none is perfect)
Example: 20 bp 50% G+C: Tm = 60 ºC
At Tm, 50% of DNA is annealed, efficient priming possible at higher
temperature.
Typical cycling parameters:
30 sec 95°C denaturation of DNA
30 sec 55°C annealing of primer
90 sec 72°C elongation (dNTP incorp.)
Slide 15
Optimising PCR specificity
Primer design:
3’ end of primer must be specific, preferably A/T G/C G/C
5’
5’ Tail does not
need to anneal
AGC3’
Template
Slide 16
Bad PCR primers
3’ overlap
Hairpin
PCR (DNA polymerase) inhibitors
• present in many impure DNA samples (blood, tissue, food etc)
> purify DNA or dilute
Slide 17
Increasing the specificity of PCR
Hot start PCR:
• Heat samples to 95 C before activating DNA polymearase
• Cool to Tm perfectly annealed primers are elongated first
Some polymerases are supplied in inactive form, e.g. bound to a specific
antibody. Incubation at 95 C removes antibody and activates polymerase.
Touch down PCR (in addition to hot start):
• Start with high annealing temperature (e.g. 65C)
• Decrease annealing temperature 1C for every cycle
Priming starts at highest possible temperature (best specificy)
cold start
1 kb
0.3 kb
Slide 18
hot start
+ touch down
Correct fragment
wrong
fragments
Increasing PCR specificity using 3’ nested primers
correct PCR fragment
wrong fragment
from unknown sequence
usually smaller than correct
fragments
Re-amplify mixture of correct and wrong PCR fragments
3’ nested
primer
correct PCR fragment
specific
Slide 19
wrong fragments are not
amplified because 3’ end of
nested primer finds no match
PCR contamination
How to prevent a single DNA molecule landing in your sample?
Serious problem where the same primer pair is used repeatedly
DNA is very stable. Soon PCR fragments are erywhere!
Disposable gloves
Filter tips
Synthesize DNA using dUTP instead of dTTP
Add heat labile Uracil-DNA Glycosylase to template
Normal DNA not affected
PCR fragments containing U are destroyed
Slide 20
Model Answers
Non-specific bands can be recognized by size, 1-primer PCR, restriction digests, reamplification using 3’-nested primers, or sequencing.
Specificity can be improved by good primers, hot start touchdown PCR, high annealing
temperature, low Mg++, enhancing agents (DMSO), not too many cycles. [I assume that
you would chose correct amounts of template, primers, dNTPs, DNA polymerase].
PCR fragments are the most common and dangerous sources of template contamination.
Clean technique (labcoat, gloves, filter tips, no aerosols (do not empty pipettes
completely; avoid DNA dust), laminar flow). Different rooms and labcoats, gloves for PCR
setup and amplification. No template controls. dUTP instead of TTP+U-DNA
glycohydrolase or similar. Note, autoclaving does not destroy DNA!
Slide 21