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Slide 1
1. Hospital
2. PCR gel
3. Results
4. Eppendorf
5. EB gel
6. K Mullis
7. Big issue
8. PCR 1-2
9. PCR 3
10. PCR Summary
11. How much DNA?
12. How many cycles?
13. Apparatus
14. Taq, PFU
15. Glossary
16. Cycling parameters
17. 3’ end for specificity
18. Bad primers
19. Hot start
20. Nested primers
21. Alder
Contents
Slide 2
Interpretation of DNA testsThere 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 3
Agarose gel for separating DNA fragments according to size
Allelic ladder
Allelic ladder
Allelic ladderM
othe
r
Fat
her
Boys1 2 3 4 5
large DNAfragments
small DNAfragments
- Migration of D
NA
+
Which boy belongs to the mother and father?
Slide 4
Allelic ladder
Allelic ladder
Allelic ladderM
othe
r
Fat
her
Boys1 2 3 4 5
Slide 5
PCRPolymerase Chain Reaction
Kary B Mullis
Method for amplifying DNA fragments
• In vitro alternative to DNA cloning
Slide 6
Day 0Investment
Day 1Profit
Double investment every day
10 days
20 days
30 days
40 days
210 = £ 1024 (103)
220 = £ 1 million (106)
230 = £ 1 billion (109)
240 = 1 trillion 1012
Slide 7
ss DNA, “Template”3’ known sequence
DNA polymerasedNTPsPrimer 1
5’
Primer 2
known sequence
95 C
Primers 1+2DNA polymerase
dNTPs
(to denature DNA)
2 partially double-stranded DNA molecules
PCR reactions 1 and 2
Slide 8
PCR fragmentdsDNA, specific lenght
partially ds DNA
95 C
Primers 1+2DNA polymerase
dNTPs
Slide 9
2 dsDNA PCR fragmentsof specific length
Primers 1+2dNTPs
3x 95 C denaturation3x DNA polymerase
dsDNATemplate
Summary PCR reactions 1-3
From now on...Every denaturation-polymerisation cycle (i) doubles the number (n) of PCR fragments
n = 2 i-2 (+2 i-3)
…………. until dNTPs or primers run out, or the enzyme activity becomes limiting.
n = 2 i-2 (+2 i-3)
Slide 10
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
How many PCR cycles starting from a single template molecule ?
40+2 cycles (c. 4 h in PCR machine)
- sufficient for 10 strong bands on agarose gel- sufficient for several cloning experiments
Slide 11
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 12
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 Contig 2Gap <10 kb
PCR
Slide 13
Yellowstone
Deep sea vent
Heat-stable DNA polymerases for PCR
Elongation at 72 °C
Survive 95 °C100 x slower than PolIII. Allow c. 1 min/kb for PCR
Taq (Thermus aquaticus) polymerase:
no proofreading
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 14
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 15
PCR primer specificityPCR 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 16
Optimising PCR specificity
Primer design:3’ end of primer must be specific, preferably A/T G/C G/C
AGC3’
5’
5’ Tail does not need to anneal
Template
Slide 17
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 18
Increasing the specificity of PCR
Correct fragment
wrongfragments
cold start hot start + touch down
1 kb
0.3 kb
Touch down PCR (in addition to hot start):
• Start with high annealing temperature (e.g. 65C)
• Decrease annealing temperature 1C for every cycle
Priming starts at highest possible temperature (best specificy)
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.
Slide 19
correct PCR fragment
Increasing PCR specificity using 3’ nested primers
wrong fragmentfrom unknown sequence
usually smaller than correct fragments
correct PCR fragment
Re-amplify mixture of correct and wrong PCR fragments
specific
3’ nested primer
wrong fragments are not amplified because 3’ end of nested primer finds no match
Slide 20
PCR contaminationHow to prevent a single DNA molecule landing in your sample?Serious problem where the same primer pair is used repeatedlyDNA is very stable. Soon PCR fragments are erywhere!
Disposable gloves
Filter tips
Synthesize DNA using dUTP instead of dTTPAdd heat labile Uracil-DNA Glycosylase to templateNormal DNA not affected PCR fragments containing U are destroyed
Slide 21
Model Answers
Non-specific bands can be recognized by size, 1-primer PCR, restriction digests, re-
amplification 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!