Slide 1 1.HospitalHospital 2.PCR gelPCR gel 3.ResultsResults 4.EppendorfEppendorf 5.EB gelEB gel 6.K MullisK Mullis 7.Big issueBig issue 8.PCR 1-2PCR 1-2.

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!