Biomarkers are pieces of DNA associated with traits in organisms. There are several types of markers including morphological, biochemical, chromosomal, and genetic. Molecular markers are preferred due to advantages like co-dominant expression, high polymorphism, and random distribution throughout the genome. Various molecular marker techniques exist like RFLP, AFLP, microsatellites, and SNPs. Marker applications include gene mapping, disease diagnosis, evolution studies, animal breeding through techniques like parentage determination, genetic distance estimation, and marker assisted selection. Overall, genetic markers provide powerful tools for the improvement and management of animal genetics.

1. BIOMARKERS
Dr .Kalaiselvigovindan
Animal biotechnology
India

2. What isWhat is
Marker?Marker?
Marker is a piece ofMarker is a piece of
DNA molecule that isDNA molecule that is
associated with a certainassociated with a certain
trait of a organismtrait of a organism
MorphologicalMorphological
BiochemicalBiochemical
ChromosomalChromosomal
GeneticGeneticTypes ofTypes of
MarkersMarkers

3. Types of biomarkerTypes of biomarker
analysisanalysis

4. Genetic evaluationGenetic evaluation

5. Animals are selected based on appearanceAnimals are selected based on appearance
Eg. PIGMENTATIONEg. PIGMENTATION
Disadvantage: lack of polymorphismDisadvantage: lack of polymorphism

6. Animals are selected based on biochemicalAnimals are selected based on biochemical
propertiesproperties
Eg. Hb, AMYLASE, BLOOD GROUPS ETC.Eg. Hb, AMYLASE, BLOOD GROUPS ETC.
Disadvantage:Disadvantage:
Sex limitedSex limited
Age dependentAge dependent
Influenced by environmentInfluenced by environment
It covers less than 10% of genomeIt covers less than 10% of genome

7. Animals are selected based on structural &Animals are selected based on structural &
numerical variationsnumerical variations
Eg. Structural and Numerical VariationsEg. Structural and Numerical Variations
Structural-Structural- Deletions, Insertions etc.Deletions, Insertions etc.
Numerical-Numerical- Trisomy, Monosomy, NullysomyTrisomy, Monosomy, Nullysomy
Disadvantage: low polymorphismDisadvantage: low polymorphism

8. Molecular MarkerMolecular Marker
 Characteristics:Characteristics:
 Co-dominant expressionCo-dominant expression
 Nondestructive assayNondestructive assay
 Complete penetranceComplete penetrance
 Early onset of phenotypicEarly onset of phenotypic
expressionexpression
 High polymorphismHigh polymorphism
 Random distributionRandom distribution
throughout the genomethroughout the genome
 Assay can be automatedAssay can be automated

9. DNA isolated from any tissue eg. Blood, hair etc.
DNA isolated at any stage even during foetal life
DNA has longer shelf-life readily exchangeable b/w labs
Analysis of DNA carried out at early age/ even at the
embryonic
Stage irrespective of sex.

10. Molecular MarkersMolecular Markers
Single locus markerSingle locus marker
Multi-locus markerMulti-locus marker
RFLP
Microsatellite
STS
DNA Fingerprinting
AFLP
RAPD

11. Randomly AmplifiedRandomly Amplified
Polymorphic DNA (RAPD)Polymorphic DNA (RAPD)

PCR based marker with 10-12 base pairsPCR based marker with 10-12 base pairs

Random amplification of severalRandom amplification of several
fragmentsfragments

Amplified fragments run in agarose gelAmplified fragments run in agarose gel
detected by EtBrdetected by EtBr

Unstable amplification leads to poorUnstable amplification leads to poor
repeatabilityrepeatability

13. Restriction FragmentRestriction Fragment
Length PolymorphismLength Polymorphism
(RFLP)(RFLP)
 Genomic DNA digested with RestrictionGenomic DNA digested with Restriction
EnzymesEnzymes
 DNA fragments separated viaDNA fragments separated via
electrophoresis and transfer to nylonelectrophoresis and transfer to nylon
membranemembrane
 Membranes exposed to probes labelledMembranes exposed to probes labelled
with Pwith P3232
via southern hybridizationvia southern hybridization
 Film exposed to X-RayFilm exposed to X-Ray

15. Amplified Fragment LengthAmplified Fragment Length
Polymorphism (AFLP)Polymorphism (AFLP)
 Restriction endonuclease digestion ofRestriction endonuclease digestion of
DNADNA
 Ligation of adaptorsLigation of adaptors
 Amplification of ligated fragmentsAmplification of ligated fragments
 Separation of the amplified fragmentsSeparation of the amplified fragments
via electrophoresis and visualizationvia electrophoresis and visualization
 AFLPs have stable amplification andAFLPs have stable amplification and
good repeatabilitygood repeatability

17. SSR: Simple SequenceSSR: Simple Sequence
Repeat or MicrosatelliteRepeat or Microsatellite
 PCR based markers with 18-25 basePCR based markers with 18-25 base
pair primerspair primers
 SSR polymorphisms are based on no.SSR polymorphisms are based on no.
of repeat units and are hypervariableof repeat units and are hypervariable
 SSRs have stable amplification andSSRs have stable amplification and
good repeatabilitygood repeatability
 SSR are easy to run and automateSSR are easy to run and automate

19. DFP: DNA finger
printing
 DNA extraction from individualDNA extraction from individual
 Amplification of markersAmplification of markers
 Electrophoresis separation of markersElectrophoresis separation of markers
 Visualization of markersVisualization of markers
 Scoring of markers for each individualScoring of markers for each individual
 Data analysisData analysis

21. FeaturesFeatures RFLPRFLP PCR-PCR-
RFLPRFLP
DFPDFP RAPDRAPD MicrosatelliteMicrosatellite SNPSNP
Detection methodDetection method HybridizationHybridization PCRPCR HybridizationHybridization PCRPCR PCRPCR PCRPCR
Type ofType of
probe/primerprobe/primer
usedused
g DNA/g DNA/
cDNA sequence ofcDNA sequence of
structural genesstructural genes
SequenceSequence
specificspecific
primersprimers
Mini satelliteMini satellite
syntheticsynthetic
oligosoligos
ArbitrarilyArbitrarily
designdesign
primerprimer
SequenceSequence
specific primersspecific primers
SequenceSequence
specificspecific
primersprimers
Requirement ofRequirement of
radioactivityradioactivity
YesYes No/YesNo/Yes YesYes No/YesNo/Yes No/YesNo/Yes No/YesNo/Yes
Extant of genomicExtant of genomic
coveragecoverage
LimitedLimited LimitedLimited ExtensiveExtensive ExtensiveExtensive ExtensiveExtensive ExtensiveExtensive
Degree ofDegree of
polymorphismspolymorphisms
LowLow LowLow HighHigh Medium toMedium to
HighHigh
HighHigh HighHigh
PhenotypePhenotype
expressionexpression
Co dominantCo dominant CoCo
dominantdominant
Co dominantCo dominant CoCo
dominant/Ddominant/D
ominantominant
DominantDominant CoCo
dominantdominant
Possibility ofPossibility of
automationautomation
NoNo YesYes NoNo YesYes YesYes YesYes

22.  Gene mappingGene mapping
 Pre and post natal diagnosisPre and post natal diagnosis
of diseasesof diseases
 Anthropological andAnthropological and
molecular evolution studiesmolecular evolution studies
Application

23. Animal breedingAnimal breeding
A.A. Conventional breeding strategiesConventional breeding strategies
1.1. Short rangeShort range
2.2. Long rangeLong range
B.B. Transgenic breeding strategiesTransgenic breeding strategies

24. Short Range ApplicationShort Range Application
 Parentage determinationParentage determination
 Genetic distance estimationGenetic distance estimation
 Determination of twin zygosity &Determination of twin zygosity &
freemartinsfreemartins
 Sexing of pre-implanted embryosSexing of pre-implanted embryos
 Identification of disease carriesIdentification of disease carries

25. Long Range ApplicationsLong Range Applications
 Gene mapping & mapping ofGene mapping & mapping of
QTL by linkageQTL by linkage
 Marker assisted selectionMarker assisted selection

26. TRANSGENIC BREEDINGTRANSGENIC BREEDING
STRATEGIESSTRATEGIES
 IDENTIFICATION OF ANIMALSIDENTIFICATION OF ANIMALS
CARRYING THE TRANSGENESCARRYING THE TRANSGENES

27. CONCLUSIONSCONCLUSIONS
The genetic improvement of animals is a continuous andThe genetic improvement of animals is a continuous and
complex process. Ever since the domestication of animalscomplex process. Ever since the domestication of animals
by man, he has always remained busy in improving hisby man, he has always remained busy in improving his
animals. In this pursuit many methods have been developedanimals. In this pursuit many methods have been developed
and tested. In recent years, the demonstration of geneticand tested. In recent years, the demonstration of genetic
polymorphism at the DNA sequence level has provided apolymorphism at the DNA sequence level has provided a
large number of marker techniques with variety oflarge number of marker techniques with variety of
applications. This has, in turn, prompted furtherapplications. This has, in turn, prompted further
consideration for the potential utility of these markers inconsideration for the potential utility of these markers in
animal breeding. However, utilization of marker-basedanimal breeding. However, utilization of marker-based
information for genetic improvement depends on the choiceinformation for genetic improvement depends on the choice
of an appropriate marker system for a given application.of an appropriate marker system for a given application.