MOLECULAR TECHNIQUESMOLECULAR TECHNIQUES

Restriction Sites, RAPDs, AFLPs & MicrosatellitesAFLPs, & Microsatellites

Faisal Ali Anwarali Khan

Molecular TechniquesMolecular Techniques

Perhaps nowhere has the power of the scientific method has been more brilliantlyscientific method has been more brilliantly demonstrated than in the development of procedures for the study of the chemistry of lifeprocedures for the study of the chemistry of life

M. O. Dayhoff and R. V. ECK (1968)

OUTLINEOUTLINE1.1. Genetic Marker Genetic Marker

N l GN l G2.2. Molecular MarkersMolecular Markers

Nuclear GeneNuclear GeneMitochondrial DNAMitochondrial DNAChloroplast DNAChloroplast DNA

3.3. Restriction sites → RFLPRestriction sites → RFLP

Chloroplast DNAChloroplast DNA

HistoryHistory4.4. RAPDRAPD

55 AFLPAFLP

HistoryHistoryMethodMethodAdvantages/Advantages/DisDis

5.5. AFLPAFLP

6.6. MicrosatellitesMicrosatellites

ApplicationApplication

7.7. Data analysis Data analysis –– BRIEFLY (AFLP & BRIEFLY (AFLP & MicrosatsMicrosats))

Genetic Marker - DescriptionGenetic Marker Description

Recognizes the characteristics of the phenotype and/orRecognizes the characteristics of the phenotype and/orRecognizes the characteristics of the phenotype and/or Recognizes the characteristics of the phenotype and/or genotype of particular individualgenotype of particular individual

Measurable characters Measurable characters –– e.g. Seed size, disease resistancee.g. Seed size, disease resistance

Their inheritance can be followed through generationsTheir inheritance can be followed through generationsTheir inheritance can be followed through generationsTheir inheritance can be followed through generations

Mendel TheoryMendel Theory

IPGRI and Cornell University, 2003IPGRI and Cornell University, 2003http://anthro.palomar.edu/mendel/mendel_1.htmhttp://anthro.palomar.edu/mendel/mendel_1.htm

Genetic MarkerGenetic MarkerMorphological traitsMorphological traits 1.1. Direct measurement of phenotypeDirect measurement of phenotypep gp g

2.2. Subject to environmental changesSubject to environmental changes3.3. EpistaticEpistatic & & PleiotropicPleiotropic

Protein markers Protein markers (biochemical markers)(biochemical markers)

1.1. Based on the Based on the migrationalmigrational properties properties of proteinsof proteins

2.2. Subject to environmental changesSubject to environmental changes3.3. Depends on developmental stagesDepends on developmental stages

•• AllozymeAllozyme

•• HistochemicalHistochemical assayassay

DNA (molecular) MarkersDNA (molecular) MarkersPCR basePCR base••NuclearNuclearNonNon--PCR basePCR base

NuclearNuclear

••MitochondrialMitochondrial

••ChloroplastChloroplast

Nuclear DNANuclear DNA

1.1. Meiosis (Recombination)Meiosis (Recombination)

Gene density and type of geneGene density and type of gene

( )( )

2.2. Mitosis process (Replication)Mitosis process (Replication)

3.3. TransposonsTransposons & CSSR& CSSRMechanism that alter Mechanism that alter 

nuclear genenuclear gene4.4. IntronIntron gain & gain & IntronIntron lossloss

5.5. DNA repair etc.DNA repair etc.Copyright © 2004 Pearson Education, Inc., publishing as Benjamin CummingsCopyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings

Mitochondrial DNAMitochondrial DNA1.1. Covalently closed duplex Covalently closed duplex

circular DNAcircular DNAcircular DNAcircular DNA

2.2. Maternally inheritedMaternally inherited

33 Non recombiningNon recombining3.3. Non recombiningNon recombining

4.4. Fast evolving Fast evolving –– different different gene different rate, higher gene different rate, higher g , gg , gthan nuclear genethan nuclear gene

5. Genealogical relationships d ti t diand estimate divergence

time

2 ribosomal RNA gene (2 ribosomal RNA gene (rRNArRNA) 22 transfer RNA) 22 transfer RNA2 ribosomal RNA gene (2 ribosomal RNA gene (rRNArRNA), 22 transfer RNA ), 22 transfer RNA gene (gene (tRNAtRNA), 13 protein genes code electron  ), 13 protein genes code electron  transportation or ATP synthesis, Control region transportation or ATP synthesis, Control region contain displacement loop (dcontain displacement loop (d‐‐loop) loop) –– in animalin animal

Chloroplast DNAChloroplast DNA1. Typical size 120-220 kb

2. 10 times larger than mtDNA

3. Circular DNA

4. Higher plants –uniparentally inherited

5. Good to compare among species then within4 rRNA genes, 30 tRNAs, 90 protein‐coding 

genes, 20 of which code for photosynthesis d l t t t f ti

6. Can be used to study geneflow

and electron‐transport functions 

Molecular TechniquesMolecular Techniques

PCR Base Technique Non PCR Base Techniqueq

RAPD=Random amplified polymorphic DNAAFLP=Amplified fragment length polymorphism

SSR/Microsatelite=Simple sequence repeats

q

RFLP=Restriction fragment length polymorphism

SSCP=Single strand conformationp q pSNP=Single nucleotide polymorphism

CAPS=Cleaved amplified polymorphic sequenceSCAR=Sequence characterized amplified region

RAMP=Randomly amplified microsatellite

SSCP=Single strand conformation polymorphism

y ppolymorphisms

DAF=DNA amplification fingerprintingSSCP=Single strand conformation

polymorphismp y pTRAP=Target region amplification polymorphism

SRAP=Sequence-related amplified polymorphism Etc.

Historical Time Frame

RFLP technique for fingerprintingRFLP technique for fingerprintingB t iB t i t l (1980) W d Whit (1980)t l (1980) W d Whit (1980)SSCP technique based on differential mobilitySSCP technique based on differential mobility

Identified in 1984 by Identified in 1984 by TautzTautz and and RentzRentz (hybridization (hybridization technique)technique)Microsatellite marker including Chloroplast and MtDNAMicrosatellite marker including Chloroplast and MtDNABosteinBostein et al. (1980); Wyman and White (1980) et al. (1980); Wyman and White (1980)

Use human samplesUse human samplesSSCP technique based on differential mobility SSCP technique based on differential mobility analysis of single stranded DNA analysis of single stranded DNA –– OritaOrita et al. (1989)et al. (1989)

Microsatellite marker including Chloroplast and MtDNA Microsatellite marker including Chloroplast and MtDNA for species specific fingerprints for species specific fingerprints –– (1992)(1992)Wentz et al. (1998) Wentz et al. (1998) -- use capillaryuse capillary

Utilization of ESTs by TRAP Utilization of ESTs by TRAP technique to detect polymorphism technique to detect polymorphism

1980 1987 19891990

------1997

------2000

------1980 1987 19891995 1999 2006

PCR technology (1987) PCR technology (1987) -- Mullis and Mullis and FaloonaFaloona ((CetusCetus Corporation) Corporation) Used not stable DNA polymeraseUsed not stable DNA polymerase

Use of arbitrary primers for PCR: RAPD, AFLP, APUse of arbitrary primers for PCR: RAPD, AFLP, AP--PCR, DAFPCR, DAFRAPD RAPD –– William et al. (1990), William et al. (1990),

use different vegetables plantuse different vegetables plant

Allelic specific Markers such as Allelic specific Markers such as CAPS & CAPS & dCAPSdCAPS to detect to detect restriction site based differencesrestriction site based differences

Employment of Employment of retrotransposonretrotransposonbase techniques such as Sbase techniques such as S--SAP, SAP, IRAP and REMAP to detectIRAP and REMAP to detectp yp y

Saiki et al. (1988) Saiki et al. (1988) –– from Henry from Henry ErlichErlich LabLabUsed stable DNA polymeraseUsed stable DNA polymerase

use different vegetables, plantuse different vegetables, plantAFLP AFLP –– Patent own by Patent own by KeygeneKeygene N. V N. V –– ZabeauZabeau and and VosVos (1993)(1993)

VosVos et al. (1995) et al. (1995) –– review on AFLP, extension of RFLPreview on AFLP, extension of RFLP

restriction site based differences restriction site based differences in in ampliconsamplicons

IRAP and REMAP to detect IRAP and REMAP to detect genome wide polymorphismgenome wide polymorphism

Modified from Agarwal et al. 2008

RFLPRFLP

http://www.familyhelix.com/articles/testing-dna/rflp-analysis.php

Advantages - DisadvantagesAdvantages DisadvantagesDNA fragment profile due to nucleotide substitution, and rearrangements

Highly polymorphic

Codominantly inheritedy

Highly reproducible

Can conduct a batch of RFLP at one time

Fig7: Diagram of an autograph

Major drawbackUse radioisotope

showing Mycobacterium tuberculosis genome

http://www3.ntu.edu.sg/home2004/WONG0172/RFLP.html

ApplicationsApplications• Intraspecific level or among closely related taxap g y

• Presence and absence of fragments resulting from changes in recognition sites are used for identifying species orrecognition sites are used for identifying species or populations

• Estimating genetic distance and fingerprinting

• Forensic biological parentage paternity cases• Forensic - biological parentage, paternity cases

• Disease status

• Genetic mapping

RAPDRAPD

Individual 1 ProductAB

Individual 2

http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/rapd.html

Advantages - DisadvantagesDNA polymorphisms –rearrangements at or between

Advantages Disadvantagesrearrangements at or between oligonucleotide primer binding sites in the genomeNo prior knowledge can beNo prior knowledge - can be employed across species using universal primersFast

Major drawbackMajor drawbacko Profiling is dependent on the

reaction conditionso Profiles are not able to distinguish

RAPD profiles for 48 samples amplified with primers OPX-06, OPX-04 and OP-AM14. In these profiles, it is possible to observe the high o Profiles are not able to distinguish

heterozygous from homozygous individuals – dominant marker

p , p greproducibility among the four different body parts from each bee (Pascual et al. 2006)

ApplicationsApplications• Characterization, estimation of genetic relatedness and g

determination of genetic diversity: Plant and animal breeding

• Able to distinguish between genotypes but limited to• 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

AFLPAFLP

https://www.msu.edu/course/mmg/835/snapshot.afs/DNAmarkers/aflp.jpg

http://www.evolutionresearchnews.org/poster.html

AdvantagesAdvantages1. Fingerprinting technique replacing RFLP1. 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

DisadvantagesDisadvantages• Dominant – lose the codominant character

• Homology – ability to differentiate different fragment with similar sizesimilar 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 inaccuratereconstruction programmes are increasingly inaccurate

– if levels of variation are high - Homoplasy

• Scoring - bias

Applications1. Monitoring inheritance of agronomic traits

Applications

2. Diagnostic in genetically inherited disease

3. Pedigree analysis,

4. Forensic typing - Parentage analysis

5 Identifying hybrids5. Identifying hybrids

6. Species level relationship

7. Also in some case at higher level relationship

Data AnalysisData Analysis• Convert AFLP profiles into binary data matrixp y

• Analyzed:

– SimilarityDistance Measures

– FrequencyDistance Measures

– Character measures

Robinson and Harris (1999); Avise 2004, Felsenstein (2004)

SimilaritySimilarity• Simple matching coefficient (Sneath and Sokal, 1973): p g ( )

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 amplified in one sample being amplified in another g p p g psample (biological perspective: inherited from a common ancestor

• Major problem: False positive – similar in RAPDRobinson and Harris (1999); Avise 2004, Felsenstein (2004)

FrequencyFrequency• The levels and patterns of diversity are calculated through p y g

frequency of a AFLP band.

• Bands are treated as independent and diversities are• 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 MeasuresCharacter Measures• Seems to be the ideal method

• Maximum likelihood – model the processes of gain or loss of AFLP b d d i lik lih d b biliti t thAFLP bands and assign likelihoods or probabilities to these events (not yet available)

• Maximum parsimony – Wagner parsimony (free reversibility) and Dollo parsimony (allows reversible once) - Which one good? Depend on the degree of divergencegood? Depend on the degree of 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 their locationTo be used as markers, their location in the genome of interest must first be identified

Polymorphisms in the repeat region can be detected by performing a PCR y p gwith 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

Si l i t t ti f lt• Simple interpretation of results

• Easily automated, allowing multiplexingEasily automated, allowing multiplexing

• Good analytical resolution and high reproducibility

• Codominant marker (“New allozyme”)

Disadvantages• Practical problems

Disadvantages

• Screening for SSR: Complex discovery procedure• Costly• Slippage: due to TaqSlippage: due to Taq• Inaccurate allele identification both in gel and

automated seq

• Data problem• Homology – the greatest problemHomology the greatest problem• Null alleles – cant amplify (sometime due to

heterozygote) (Dakin and Avise 2004)

Robinson and Harris (1999)

ApplicationsApplicationsIndividual genotypingIndividual genotyping

Parentage

Genetic diversity, population genetic study

Genome mapping

Evolutionary studies - Hybridization

Data AnalysisData Analysis

• Can be analyzed:

b f ll l h d– Presence or absence of alleles as characters, and 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/AbsencePresent/Absence• Argued as invalid method:g

– Independent losses of "primitive" alleles = synapomorphies– Loci with > number of alleles = weight > in tree

reconstructionreconstruction– 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 butanalyzed 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 phylogeneticsin phylogenetics

– Question whether model of repeat evolution should be the infinite allele model (no reverse) or stepwise mutation model (only gain or lose onemodel (no reverse) or stepwise mutation model (only gain or lose onerepeat)

• 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 MarkerIdeal Molecular Marker

SORRY WE DON’T HAVE ONE YET!!