The polymerase chain reaction (PCR) can produce many copies of a specific segment of DNA. It works through a three-step cycle of heating, cooling, and replication that causes exponential growth in the number of DNA molecules matching the target sequence. PCR is a versatile technique for amplifying DNA sequences in vitro. It is sensitive, quick, easy to use, and robust.

1. LECTURE 5
Polymerase Chain Reaction

2. Amplifying DNA in Vitro: The
Polymerase Chain Reaction (PCR)
 The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
 A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population
of identical DNA molecules

3. Polymerase chain reaction
 a.k.a. DNA amplification
 in vitro method to specifically amplify nucleic acid
sequences
 A most important and versatile technique




Very sensitive
Quick
Easy
robust
http://highered.mcgraw-hill.com/sites/0073031208/

4. Amplification by replicating
a specific sequence of DNA
many times
From one…
…to billions
of copies
Brock Biology of the Microorganism

5. Fig. 20-8
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
5
5
3
3
1 Denaturation
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence

6. Fig. 20-8a
5
TECHNIQUE
3
Target
sequence
Genomic DNA
3
5

7. Fig. 20-8b
5
3
3
1 Denaturation
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides

8. Fig. 20-8c
Cycle 2
yields
4
molecules

9. Fig. 20-8d
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence

10. DNA Amplification = DNA replication
Same requirements !
Double helix must unwind
Primers
Deoxynucleotides (dNTPs)
Polymerase
Brock Biology of the Microorganism

11. allserv.rug.ac.be/~avierstr/ principles/pcr.html

12. http://oceanexplorer.noaa.gov/explorations/04etta/background/dna/dna_1_220.jpg

13. Brock Biology of the Microorganism

14. http://www.agen.ufl.edu/~chyn/age2062/lect/lect_09/lect_09.htm

15. Important to understand :
 PCR is specific
 because of the primers
 we design the primers to anneal to specific sequences
 That means, we must know what we want to amplify
 The sequence between the two primers is amplified
 other parts of the DNA will not be amplified
 Amplification is exponential
 the newly synthesised DNA served as templates for further
rounds of amplification

16. The power of PCR
 e.g. detection of viral /bacterial pathogens
 Current technology




-
-
culture the pathogen
Run biochemical/ immunological test
Time consuming
Some viruses & bacteria cannot yet be cultured
Delay diagnosis, wrong diagnosis, or not specific
enough
e.g. SARS, bird flu

17. With PCR
 Go straight for the DNA
 No need to culture, biochemical test etc.
 e.g Bacillus anthracis
 First, we must be able to identify a gene/DNA sequence that is
unique to the anthrax bacteria
 This gene or sequence must be present only in the B.anthracis
DNA, and not the DNA of any other organism, virus etc.

18.  Then, we need to know a bit of the DNA sequence of this
anthrax gene (the ‘target’)
 So that we can design specific primers that only anneals
to the target anthrax DNA
 and not any other DNA

19. Then we do a PCR using these anthrax-specific primers
and DNA extracted from patient’s blood
Only a very small amount of blood is required < 0.1 ml
http://nobelprize.org/chemistry/laureates/1993/illpres/pcr.gif

20. +ve sample
Primers
anneal to
target DNA
anthrax
gene is
amplified
-ve sample
No amplification
Because
primers cannot
anneal with any
other DNA

21. Check for amplification products using a gel

22. PCR offers many advantages :
 no need to isolate/culture the pathogens – extract DNA from soil and




other samples
Very sensitive – amplify from only one copy of target (well, in
theory), so requires very little sample
Highly specific – can even identify different species or strains
accurately
Fast – a few hours
Allows for diagnosis before disease develops
 This is only one of the many applications of PCR, we will discuss
may more later

23. What I need ?
How to do
a PCR ?

24. PCR reaction requires
 Target DNA – a tiny bit will do
 Specific primers - most crucial


Must know sequence
Must be correctly designed
 DNA polymerase - heat stable
 Thermocycler - machine to heat
and cool
 dNTPs – building blocks for new
DNA synthesis
 Buffer – to provide correct conditions
for the enzyme to work

25.  Typical reaction mix
 Buffer + MgCl2
 dNTPs – dATP,dCTP,dTTP,dGTP
 Primers - forward and reverse
 Polymerase
 DNA sample
 Thermocycling
 Denaturation 94C
 Annealing
55 C
 Extension
72C

26. Buffer & MgCl2
 Always use buffer that comes with enzyme
(unless you’re one of those who knows better!)
 MgCl2 - affects specificity and yield
 usually about 2 mM
 High [MgCl] - more product but less specific
 Low [MgCl] – less product but more specific
 Optimise !

27. dNTPs
 premade & premix – just buy them
 Use 50 to 500 mM each
 50 mM enough to make 6 ug of products

28. Primers




Most crucial
Primers are designed by you and synthesized on a machine
Occasionally primers fail for apparently no reason, so don’t feel bad
Ensure quality of primers – get a good supplier
Guidelines
 Check orientation of primers
 20 to 30 base pair long
 Go for 40 to 50% GC content
 Avoid internal structure
 Avoid complimentarity between primers, esp at 3’ end
 Avoid extensive GC’s at 3’ end

29. Orientation of primers
CTTATTAGTTTACTAT
5’CTTATTAGTTTACTATAAAGGAGTCGAAAGAGAAGTACCAAAGAT 3’
3’GAATAATCAAATGATATTTCCTCAGCTTTCTCTTCATGGTTTCTA 5’
.
CTCTTCATGGTTTCTA
.
Forward primer = 5’CTTATTAGTTTACTAT 3’
Reverse primer = 5’ATCTTTGGTACTTCTC 3’
5’CTTATTAGTTTACTATAAAGGAGTCGAAAGAGAAGTACCAAAGAT 3’
3’CTCTTCATGGTTTCTA 5’
5’CTTATTAGTTTACTAT 3’
3’GAATAATCAAATGATATTTCCTCAGCTTTCTCTTCATGGTTTCTA 5’

30.  Correct primers  amplification
 One wrong primer  no amplification
 Two wrong primers  no amplification

31. Things to avoid

Internal structures
cattgccgacggcttaatcgta
a
g=c
c=g
c=g
cattg=cttaatcgta

a ‘loop out’
Complementary 3’ ends
5’ cgtacgtactggttacctacgc 3’
‘primer dimer’
| | | | | | |
3’ ggatgcgaattagactgacgc 5’

32. Polymerase
 Taq polymerase from Thermus aquaticus
 thermophilic - works at 72C
 Others – Vent, Deepvent, TaKaRa, etc.

33. Polymerase makes error !
Brock Biology of the Microorganism

34.  Taq – no proof-reading activity – doesn’t correct error
 Also add an extra base – A – at the ends

A

A
 Error rate can be as high as one mistake in 1000
 New generation of ‘proof reading enzymes’ –


e.g. Pfu, Pfx, Pwo
has 3’exonuclease and proofreading activity
 Extra A at the end – can be used in T/A cloning systems

35. Applications of PCR
 Medical
 Forensics (CSI)
 Detection of infectious agents



Viral infections
Bioweapons
Difficult-to-culture organism or slow growing

36. Forensic Evidence and Genetic
Profiles
 An individual’s unique DNA sequence, or genetic
profile, can be obtained by analysis of tissue or body
fluids
 Genetic profiles can be used to provide evidence in
criminal and paternity cases and to identify human
remains
 Genetic profiles can be analyzed using RFLP analysis
by Southern blotting

37.  Even more sensitive is the use of genetic
markers called short tandem repeats (STRs),
which are variations in the number of repeats
of specific DNA sequences
 PCR and gel electrophoresis are used to
amplify and then identify STRs of different
lengths
 The probability that two people who are not
identical twins have the same STR markers is
exceptionally small

38. Fig. 20-24
(a) This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
STR
marker 2
STR
marker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b) These and other STR data exonerated Washington and
led Tinsley to plead guilty to the murder.

39. Time to watch a movie….

40. Reverse Transcriptase PCR for the detection of RNA viruses
(almost all of the most nasty viruses have RNA genomes –Ebola,
dengue, nipah, SARS – you name it)
- before PCR – reverse transcribed viral RNA to cDNA first
Brock Biology of the Microorganism

41. DNA fingerprinting

42. DNA fingerprinting using VNTRs
http://homepages.strath.ac.uk/~dfs97113/BB310/Lect1603.html

43. VNTRs - variable number of tandem repeats – a.k.a minisatellites
– natural polymorphisms in the human genome
Different numbers of a short, repeated sequence
Each repeat 15 – 100 bp long;
Repeated in tandem arrays up to 40 kb long
SSTR – simple sequence tandem repeats – a.k.a microsatellites
Repeats of 2 to 4 nucleotides
e.g.
CAGCAGCAGCAGCAGCAGCAG
daddy
CAGCAGCAGCAG
mommy
daddy
mommy
VNTRs are hypervariable – can be very different between individuals

44. Many different types of VNTRs
-can be found at many loci in the genome
-two individual may have similar VNTRs at one loci
-But the chances of two individuals having the same pattern of VNTRs
at several loci is very small

45. The DNA sequences next to VNTRs are usually highly conserved
(very similar in every individual)
So we can design PCR primer to target these flanking sequences
Using these primers, we can amplify the VNTR regions
The VNTR amplification products will have different sizes, and can
be separated on an agarose or polyacrylamide gel

46. By using PCR to amplify all three regions, a unique
fingerprint can be generated for each individual

48. Fingerprinting by PCR of VNTR/STR

49. www-ermm.cbcu.cam.ac.uk/ 99000587h.htm

50. Family trees by DNA finger printing
http://www.people.virginia.edu/~rjh9u/vntr1.html

51. DNA fingerprinting

52. DNA fingerprinting

53. Real time PCR – the next evolution
 Monitoring of amplification reaction in real time
 Quantitative
 Rapid - results-as-you –wait
 Close system – minimal cross contamination
 Much more expensive…………..

54. Detection of GMO’s
 Most genetically modified plants contain the 35S promoter of
cauliflower mosaic virus (CaMV)
 and the 3’ untranslated region (terminator) ofthe nopaline
synthase (NOS) gene of Agrobacterium tumefaciens.
 Can be detected using specific primers

55. Detection using SYBRO Green or fluorescence molecular beacons
SYBRO Green fluoresces when bind to double stranded DNA only

56. Brock Biology of the Microorganism

57. Detection of GM soya and maize by RT PCR