Molecular Biology
Materials & Methods 5:
PCR and DNA Markers
1
Kary Mullis, after winning Nobel Prize for inventing PCR
2
www.palamito.it/fotopalfiles
PCR: Polymerase Chain Reaction
• ―Amplify‖ DNA by in-vitro (in plastico)
synthesis
• Key requirements:
– enzyme: Taq DNA polymerase, not denatured
at high temps used to denature DNA
– primers: short (~ 20 b) oligonucleotides bind
to denatured DNA, required to start DNA
synthesis
3
4
5
6
7
8
9
10
11
12
PCR amplifies the
amount of DNA—
doubles each cycle,
so it increases
exponentially
13
Scientific American 14
With the addition
of thermostable
Taq polymerase,
PCR was adopted
immediately, in
many different
areas of biology.
15
16
17
PCR: polymerase chain reaction
• Advantages:
– powerful: works with miniscule amounts of DNA (even
single cells)
– highly specific targets
– many primers work with broad range of targets
(plants, animals, fungi)
– adapted to simplify DNA sequencing
• Disadvantages:
– difficulty / expense of primer development
– troubleshooting when reactions fail
18
19
20
21
Thermosequencing: dideoxy
sequencing + PCR
• Recall fundamental step of Sanger
sequencing is enzymatic replication of
DNA
• PCR using only one primer, plus different
fluorescent nucleotides for each of 4
ddNTP reactions, produces copies of one
strand
• Time-consuming steps of digestion &
cloning in M13 can be avoided
22
PCR anthem from Bio-Rad
• http://www.cnpg.com/video/redirect.aspx
?redirectid=65
(n.b. slide moved from location on your handout)
23
RFLPs: Restriction Fragment
Length Polymorphisms
• ―Restriction‖ enzymes digest DNA by
cutting only at specific sequences
(typically 6 bp palindromes)
• Named because they restrict the entry of
foreign (e.g. viral) DNA into bacteria, by
shredding it
24
25
26
27
28
29
Lespedeza capitata, L. leptostachya
and hybrid
mitochondrial DNA
digested with Eco RI
probed with cox III
plant mtDNA is inherited maternally
(i.e. through seed, not pollen)
Lespedeza hybrids
- suspected from morphology
-confirmed by isozymes
- mtDNA RFLPs demonstrate hybridization
occurred by pollen from capitata
onto flowers of leptostachya
30
VNTRs – DNA fingerprints
• A kind of RFLP: combines restriction
digest & probing
• Detects ―variable number of tandem
repeats‖ polymorphism
31
32
33
34
35
DNA Fingerprint -- VNTR
Arrows indicate bands
present in putative father no. 2
that are:
1) present in child
2) absent in mother
3) absent in putative father no.1
Snustad & Simmons 200036
VNTR fingerprints
• Advantages:
– Low development cost: probes work on most taxa
(plants and animals)
– Highly polymorphic
• Disadvantages:
– Bands ―anonymous‖: can’t determine how many loci
or alleles are present
• Mostly forensic applications
– Comparisons among a small & defined set of related
individuals
37
RAPDs: Randomly Amplified
Polymorphic DNA
• PCR using short (10 b) primers
• Short primers bind to many sites scattered
throughout the genome
– because the
38
39
40
41
42
43
RAPDs in Aconitum
ugh
44
RAPDs: Randomly Amplified
Polymorphic DNAs
• Advantages:
– Modest development cost
– Low application cost
– High polymorphism
– Modest database for comparison
• Disadvantages:
– Bands dominant & anonymous: can’t
recognize alleles for a locus
– Generally not comparable among labs
45
AFLPs: Amplification Fragment
Length Polymorphisms
• Combines RFLPs + PCR
46
47
48
49
50
51
Owen’s AFLPs in Polygonella
52
AFLPs: Amplification Fragment
Length Polymorphisms
• Advantages:
– Moderate development cost
– Highly polymorphic
• Disadvantages
– Anonymous bands (can’t tell loci from alleles)
– Dominant (complex & uncertain genetics)
– Rather expensive (multiple restrictions plus
PCR plus capillary electrophoresis)
53
Microsatellites / SSRs:
simple sequence repeats
• PCR-based assay for very small VNTRs
• Amplify using one of:
– radiolabeled nucleotides
– fluorophore-labeled nucleotides
– fluorophore-labeled primers
54
55
56
57
SSR amplification using radiolabeled nucleotides
Cregan et al. 1994 58
Capillary electrophoresis
allows high throughput
and small reaction volumes
59
SSRs in aspens
60
SSR Advantages
• Defined loci
• Defined alleles
– capillary eph gives < 0.5 b resolution
• Robust amplification targets
• Codominant !!
• Highly polymorphic
61
SSR Disadvantages
• Primer development takes lots of work
– digest, clone, probe, sequence, design primer
• Primers not broadly applicable across taxa
– usually work within genera (plants)
• Fluorescent primers are expensive
62
Molecular beacon for detecting PCR product:
a specfic probe with a fluorophore & quencher at each end
Sequence complementary to sequence of PCR product
stems –
complementary
Fluorophore to each other,
quenched by not to PCR
proximity to product
quencher
Quencher
Hybridized fluorophore reveals presence of PCR product
63
Molecular beacons can detect single-nucleotide polymorphisms:
binding portions of probes differ by a single base
green beacon yellow beacon heterozygote
detects wild-type detects mutant has both
allele allele beacons
64
Molecular beacons improve quantitative PCR:
amount of product reflects amount of starting target
LOTS of target
not much
target
65