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MOLECULAR TECHNIQUES
Restriction Sites, RAPDs,
AFLPs & Microsatellites
AFLPs,
Faisal Ali Anwarali Khan
Molecular Techniques
Perhaps nowhere has the power of the scientific method has been more brilliantly
scientific method has been more brilliantly demonstrated than in the development of procedures for the study of the chemistry of life
procedures for the study of the chemistry of life
M. O. Dayhoff and R. V. ECK (1968)
OUTLINE
1. Genetic Marker
2. Molecular Markers
N l
Nuclear
G
Gene
Mitochondrial DNA
Chloroplast DNA
3. Restriction sites → RFLP
4. RAPD
5 AFLP
5.
History
Method
Advantages/Dis
Advantages/
Dis
Application
6. Microsatellites
7. Data analysis – BRIEFLY (AFLP & Microsats
Microsats))
Genetic Marker - Description
™ Recognizes the characteristics of the phenotype and/or
genotype of particular individual
™ Measurable characters – e.g. Seed size, disease resistance
™ Their inheritance can be followed through generations
Mendel Theory
http://anthro.palomar.edu/mendel/mendel_1.htm
IPGRI and Cornell University, 2003
Genetic Marker
ƒ Morphological
p
g
traits
1. Direct measurement of phenotype
2. Subject to environmental changes
3. Epistatic & Pleiotropic
ƒ Protein markers
(biochemical markers)
• Allozyme
• Histochemical assay
1. Based on the migrational properties
of proteins
2. Subject to environmental changes
3. Depends on developmental stages
ƒ DNA (molecular) Markers
•Nuclear
•Mitochondrial
•Chloroplast
PCR base
Non--PCR base
Non
Nuclear DNA
Gene density and type of gene
1.
2.
3.
4.
5.
Meiosis (Recombination)
(
)
Mitosis process (Replication)
Transposons & CSSR
Intron gain & gain & Intron
Intron loss
DNA repair etc.
Mechanism that alter nuclear gene
Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings
Mitochondrial DNA
1. Covalently closed duplex
circular DNA
2. Maternally inherited
3 Non recombining
3.
4. Fast evolving – different
gene different rate,, higher
g
g
than nuclear gene
5. Genealogical relationships
and
d estimate
ti t divergence
di
time
2
2 ribosomal RNA gene (
2 ribosomal RNA gene (rRNA
ribosomal RNA gene (rRNA
(rRNA), 22 transfer RNA rRNA))), 22 transfer RNA 22 transfer RNA
gene (tRNA
gene (
tRNA), 13 protein genes code electron ), 13 protein genes code electron transportation or ATP synthesis, Control region contain displacement loop (d‐‐loop) contain displacement loop (d
loop) –– in animal
Chloroplast DNA
1. Typical size 120-220 kb
2. 10 times larger than
mtDNA
3. Circular DNA
4. Higher plants –
uniparentally inherited
4 rRNA genes, 30 tRNAs, 90 protein‐coding genes, 20 of which code for photosynthesis and electron‐transport functions d l t
t
t f ti
5. Good to compare among
species then within
6. Can be used to study
geneflow
Molecular Techniques
PCR Base Technique
q
Non PCR Base Technique
q
RAPD=Random amplified polymorphic DNA
AFLP=Amplified fragment length polymorphism
SSR/Microsatelite=Simple
p sequence
q
repeats
p
SNP=Single nucleotide polymorphism
CAPS=Cleaved amplified polymorphic sequence
SCAR=Sequence characterized amplified region
RAMP=Randomlyy amplified
p
microsatellite
polymorphisms
DAF=DNA amplification fingerprinting
SSCP=Single strand conformation
p y
polymorphism
p
TRAP=Target region amplification polymorphism
SRAP=Sequence-related amplified
polymorphism Etc.
RFLP=Restriction fragment length
polymorphism
SSCP=Single strand conformation
polymorphism
Historical Time Frame
™ Identified in 1984 by Tautz and Rentz (hybridization
technique)
RFLP technique
for fingerprinting
Microsatellite
including
Chloroplast
basedand
on
mobilityand MtDNA
™ Bostein
B t™iSSCP
ett al.
ltechnique
(1980);
(1980) Wyman
Wmarker
d differential
White
Whit (1980)
™ fingerprints
Utilization
ESTs
by(1989)
TRAP
for species
specific
–of(1992)
analysis
of single
stranded
DNA – Orita
et al.
™ Use human
samples
technique
™ Wentz et al. (1998) - use
capillaryto detect polymorphism
1980
1987
1989
1990
1997
2000
------
------
------
1995
1999
2006
Use of arbitrary primers
forspecific
PCR: RAPD,
AFLP,
APas
AP-PCR, DAF
Employment
of retrotransposon
Allelic
Markers
such
PCR
technology
- base
Mullis
and Faloona
(Cetus
RAPD
– William (1987)
et al.
(1990),
techniques
such
as SS-Corporation)
SAP,
CAPS
& dCAPS
to detect
stablevegetables
DNA
polymerase
p
y plant
™
™ Used
use not
different
vegetables,
IRAP
and
to detect
restriction
siteREMAP
based differences
Saiki
(1988)
– from
Henry Erlich
Lab
AFLPet–al.
Patent
own
by
Keygene
N.
V–
Zabeau and Vos (1993)
genome
wide
polymorphism
in amplicons
™
DNA polymerase
™ Used
Vos stable
et al. (1995)
– review on AFLP, extension of RFLP
Modified from Agarwal et al. 2008
RFLP
http://www.familyhelix.com/articles/testing-dna/rflp-analysis.php
Advantages - Disadvantages
™ DNA fragment profile due to
nucleotide substitution, and
rearrangements
™ Highly polymorphic
™ Codominantlyy inherited
™ Highly reproducible
™ Can conduct a batch of RFLP
at one time
™ Major drawback
™ Use radioisotope
Fig7: Diagram of an autograph
showing Mycobacterium
tuberculosis genome
http://www3.ntu.edu.sg/home2004/WONG0172/RFLP.html
Applications
• Intraspecific
p
level or among
g closely
y related taxa
• Presence and absence of fragments resulting from changes in
recognition sites are used for identifying species or
populations
• Estimating genetic distance and fingerprinting
• Forensic - biological parentage
parentage, paternity cases
• Disease status
• Genetic mapping
RAPD
Individual 1
Product
A
B
Individual 2
http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/rapd.html
Advantages - Disadvantages
™ DNA polymorphisms –
rearrangements at or between
oligonucleotide primer binding sites
in the genome
™ No prior knowledge - can be
employed across species using
universal primers
™ Fast
™ Major drawback
o Profiling is dependent on the
reaction conditions
o Profiles are not able to distinguish
heterozygous from homozygous
individuals – dominant marker
RAPD profiles for 48 samples amplified with
primers OPX-06, OPX-04 and OP-AM14. In
these p
profiles,, it is possible
p
to observe the high
g
reproducibility among the four different body
parts from each bee (Pascual et al. 2006)
Applications
• Characterization, estimation of g
genetic relatedness and
determination of genetic diversity: Plant and animal breeding
• Able to distinguish between genotypes but limited to
comparisons of populations from a few sources –
identification marker
• Genetic mapping – drawback is dominant so need more
character
• Population and evolutionary genetics
AFLP
https://www.msu.edu/course/mmg/835/snapshot.afs/DNAmarkers/aflp.jpg
http://www.evolutionresearchnews.org/poster.html
Advantages
1. Fingerprinting technique replacing RFLP
2. Highly polymorphic
3. High reproducibility??
4. Identify through absent or present of fragment
5. Characters can be increased by changing the RE
and nucleotide at selective primers
Disadvantages
• Dominant – lose the codominant character
• Homology – ability to differentiate different fragment with
similar size
• Mutation rate – high homoplasy.
– High levels of variation - similarity between two taxa are
low, so both character and distance measures and tree
reconstruction programmes are increasingly inaccurate
– if levels of variation are high - Homoplasy
• Scoring - bias
Applications
1. Monitoring inheritance of agronomic traits
2. Diagnostic in genetically inherited disease
3. Pedigree analysis,
4. Forensic typing - Parentage analysis
5 Identifying hybrids
5.
6. Species level relationship
7. Also in some case at higher level relationship
Data Analysis
• Convert AFLP p
profiles into binary
y data matrix
• Analyzed:
– Similarity
Distance Measures
– Frequency
– Character measures
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Similarity
• Simple
p matching
g coefficient ((Sneath and Sokal, 1973):
)
measures the proportion of shared band presence and
absences
• Jaccard's coefficient (Jaccard, 1908): Proportion of shared
bands
• Nei and Li coefficient (Nei and Li, 1979): Probability a band
being
g amplified
p
in one sample
p being
g amplified
p
in another
sample (biological perspective: inherited from a common
ancestor
• Major problem: False positive – similar in RAPD
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Frequency
• The levels and p
patterns of diversity
y are calculated through
g
frequency of a AFLP band.
• Bands are treated as independent and diversities are
calculated using:
– Similarity measures
– Shannon's measure
– Analysis of molecular variance
• Major problem: False positive – similar in RAPD, additional
problem deals with dominant that increased frequency
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Character Measures
• Seems to be the ideal method
• Maximum likelihood – model the processes of gain or loss of AFLP bands and assign likelihoods or probabilities to these AFLP
b d
d i lik lih d
b biliti t th
events (not yet available)
• Maximum parsimony – Wagner parsimony (free reversibility)
and Dollo parsimony (allows reversible once) - Which one
good? Depend on the degree of divergence.
divergence
• Dollo seems to be good for AFLP because huge restriction
sites, so have asymmetry changes of gain and loss
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Microsatellite (SSR)
™ Microsatellites are short tandem
repeats (1-10 bp) – “junk” DNA
™ To be used as markers
markers, their location
in the genome of interest must first be
identified
™ Polymorphisms in the repeat region
can be detected by
y performing
p
g a PCR
with primers designed from the DNA
flanking region
IPGRI and Cornell University, 2003
http://www.geneservice.co.uk/services/faqs/
Advantages
• Require very little and not necessarily high quality DNA
• Highly polymorphic
• Evenly distributed throughout the genome
• Simple
Si l iinterpretation
t
t ti off results
lt
• Easily automated, allowing multiplexing
• Good analytical resolution and high reproducibility
• Codominant marker (“New allozyme”)
Disadvantages
• Practical problems
• Screening for SSR: Complex discovery procedure
• Costly
• Slippage: due to Taq
• Inaccurate allele identification both in gel and
automated seq
• Data problem
• Homology – the greatest problem
• Null alleles – cant amplify (sometime due to
heterozygote) (Dakin and Avise 2004)
Robinson and Harris (1999)
Applications
™ Individual genotyping
™ Parentage
™ Genetic diversity, population genetic study
™ Genome mapping
™ Evolutionary studies - Hybridization
Data Analysis
• Can be analyzed:
– Presence or absence of alleles as characters, and b
f ll l
h
d
calculating either distance or using character measures
– Allele frequency at loci as characters and calculating distance measures
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Present/Absence
• Argued
g
as invalid method:
– Independent losses of "primitive" alleles = synapomorphies
– Loci with > number of alleles = weight > in tree
reconstruction
– Character conflicts when no alleles shared between the
ingroup and outgroup
• Data may be converted into a pair-wise similarity matrix or
analyzed as character data – used in maximum parsimony but
resulted in less parsimonious relationship
• Both method biased estimating relationships (homology)
(
)
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Allele frequency
• Difficulties in data coding and computing distances – not used
in phylogenetics
– Question whether model of repeat evolution should be the infinite allele
model (no reverse) or stepwise mutation model (only gain or lose one
repeat)
• SMM consistent with the observed allele frequencies at SSR loci
– Two-phase model - in which the primary changes are single addition or
losses of repeats with the occasional rare large change in repeat
number
– Nature of allele frequencies - not temporally stable, therefore not
synapomorphic; effects of non-homologous and null alleles
Robinson and Harris (1999); Avise 2004, Felsenstein (2004)
Ideal Molecular Marker
SORRY WE DON’T HAVE ONE YET!!