this presentation is about the molecular markers as we all know the molecular markers are the DNA sequences it can be easily detected and its inheritance is easily monitored.so the main basics of the molecular markers is the polymorphic nature so it can used as molecular markers.and this will gives you the idea about AFLP, RFLP, RAPD, SNPS,ETC.

1. MOLECULAR MARKERS

2. •A DNA sequence that is readily detected and whose inheritance can be easily monitored.
•The uses of molecular markers are based on the naturally occurring polymorphism.
•A marker is a gene of known function and location, that allow the studying of the
inheritance of the gene.
•A marker must be a polymorphic i.e., it must exist in different forms so that chromosomes
carrying mutant gene can be distinguished from the chromosome with the normal gene
by a marker.
• NB: polymorphism involves existence of different forms of same gene in plants or
population of plants.
Examples: RFLP,RAPD,AFLP, VNTR,SSR,SNP etc…

3. They may be due to:
• Base pair changes.
• Rearrangements (translocation or
inversion).
• Insertions or deletions.
• Variation in the number of tandem
repeats.
THE MARKERS REVEALING VARIATION AT DNA LEVEL
ARE REFERRED TO AS THE MOLECULAR MARKERS.

4. Protein markers such as alloenzymes
DNA markers such as:
MITOCHONDRIAL DNA MARKERS
NUCLEAR DNA MARKERS ( RAPD ,
VNTR SUCH AS MICRO & MINI SATELLITES)

5. A MOLECULAR MARKER MUST POSSES THE FOLLOWING
DESIRABLE PROPERTIES
It must be polymorphic ( different structures in
different individuals of a species ) so that diversity
must be measured
It should be evenly distributed throughout the
genome
It should distinguishes the homozygotes and
heterozygotes.

6. In the organism ,the polymorphism can be distinguished
at three levels
1. Morphological or phenotypic; i.e., The morphological marker
corresponds to a quantitative trait scored visually
2. Biochemical marker due to difference in protein
e.g.; Isozymes
3. Molecular marker-DNA sequences among which the differences
can be detected and monitored in the subsequent generation

7. Advantages of Molecular Markers
 Ubiquitous.
 Stably inherited.
 Multiple alleles for each marker.
 Devoid of pleiotropic effects.
 Detectable in all tissues, at all ages.
 Long shelf life of the DNA samples.

8. RFLP – (RESTRICTION FRAGMENT LENGTH POLYMORPHISM)
•Genetic markers
resulting from the
variation or change in
the length of defined
DNA fragments
produced by digestion
of the DNA sample with
restriction
endonucleases

9. RFLP is a difference in homologous DNA sequences that can be detected by
presence of fragment of different lengths after digestion of the DNA samples
with specific restriction endonucleases.
When the genomic DNA of many individuals of one species are separately
cleaved by restriction enzymes ,passed through electrophoresis.
Then it blotted on nitrocellulose membrane and probed with a radio labelled
DNA.
There arises polymorphism in hybridisation pattern of digested DNAs
This shows differences in sequences between two individual. These unique,
common and repeated DNA sequences found in several individuals are
termed as RFLPS.
After digestion with specific enzymes, variations obtained in one DNA
fragment with that enzyme is referred to as one RFLP.

10. The RFLPs are detected by using the unique sequence of DNA called genomic probe,
RFLP map constructed as follows
Large molecular weight genomic DNA is isolated from several strains or related species
These DNAs are then digested with a selected restriction enzyme. The fragments in these
digest are separated through electrophoresis
The resulting gel lines are transferred to a suitable solid support and exposed to e radio
labelled probe under conditions favouring hybridisation
The free probes are removed and finally the fragments to which the probe has hybridised
are detected by autoradiography
An RFLP occurs when the length of a detected fragments varies between individuals
Each fragment length is considered an allele ,and can be used in genetic analysis

11. Microbial cell or plant / animal tissue
Genomic DNA
DNA fragments separated on gel
DNA on nitrocellulose membrane
Southern hybridisation
Isolate DNA
Digest DNA with restriction enzymes
Separate on agarose gel
Southern blotting
Incubate with suitable genome cloned probe
autoradiography
Positive band shows RFLP PATTERN

12. RFLP analysis
Polymorphism revealed by different
probe/enzyme combinations among 13
different accessions.

13. •High reproducibility
•Show co-dominant alleles
•Detect coupling phase of DNA
•Reliable marker in linkage and breeding analysis
•Easily determine a linked trait present in both homozygous and heterozygous
ADVANTAGES

14. •Require large quantities of high molecular weight DNA.
•Expensive process
•Time consuming
•Labor intensive
DISADVANTAGES

15. CONSIDERATIONS FOR USE OF RFLPS
-Relatively slow process
-Use of radioisotopes has limited RFLP use to
certified laboratories (but non-radioactive labeling
systems are now in wide use)
- Co-dominant markers; often species-specific
- Need high quality DNA
- Need to develop polymorphic probes - expensive

17.  AFLP is based on a selectively amplifying a subset of restriction fragments from a
complex mixture of DNA fragments obtained after digestion of genomic DNA with
restriction endonucleases.
 Polymorphisms are detected from differences in the length of the amplified
fragments by polyacrylamide gel electrophoresis (PAGE) .
 The technique involves four steps: (1) restriction of DNA and
ligation of oligonucleotide adapters (2) preselective amplification (3)
selective amplification (4) gel analysis of amplified fragments.
 AFLP involves the restriction of genomic DNA, followed by ligation of adaptors
complementary to the restriction sites and selective PCR amplification of a subset of the
adapted restriction fragments. These fragments are viewed on denaturing
polyacrylamide gels either through autoradiographic or fluorescence methodologies .
AFLP(AMPLIFIED FRAGMENT LENGTH POLYMORPHISM)

18.  High genomic abundance.
Considerable reproducibility.
 AFLPs can be analyzed on automatic sequencers.
 The generation of many informative bands per reaction.
Capability to amplify between 50 and 100 fragments at
one time.
Higher resolution and sensitivity.
ADVANTAGES

19. • Need for purified, high molecular
weight DNA.
• The major disadvantage of AFLP
markers is that these are dominant
markers.
•Abundance of data.
DISADVANTAGES

20. •AFLPs can be applied in studies involving genetic identity, parentage and
identification of clones and cultivars.
•phylogenetic studies of closely related species.
• AFLP markers have successfully been used for analyzing genetic diversity in some
other plant species such as peanut.
•This technique is useful for breeders to accelerate plant improvement.
•AFLP markers are useful in genetic studies, such as biodiversity evaluation,
analysis of germplasm collections, genotyping of individuals and genetic distance
analyses.
APPLICATIONS

21. RAPD ( RANDOM AMPLIFIED POLYMORPHIC DNA )
•It is a PCR based technology.
•In 1991 Welsh and Maclelland developed this technique.
•This procedure detects nucleotide sequence polymorphism in DNA.
•It is used to analyze genetic diversity of an individual by random primers.
• RAPD generates a type of genetic finger print that can be used to identify individuals
• If we have some information about a set of fragments randomly distributed through out the
genome ,we may synthesize a single short oligonucleotide (10bp) and use both ends of DNA
segments before its amplification
• The DNA amplification product is generate from a region flanked by a pair of 10bp primary
sites, consequently ,random sample of DNA markers is obtained which is called RAPD
• Unlike the traditional PCR analysis ,RAPD does not require any specific knowledge of the DNA
sequences of the target organisms.
• Genomic DNA of two individuals produce different RAPDs.

22. •A specific DNA segment shows DNA polymorphism. Hence it can be used as a
genetic marker
• Many random oligonucleotide primers are designed by using different
combination of nucleotides.
• This makes the method popular for comparing the DNA of biological systems that
have not had the attention of the scientific community
• It has some limitation in the use of degraded DNA samples .
• It’s resolving power is much lower than targeted ,species specific DNA
comparison methods ,such as short tandem repeats .
• In recent years ,RAPD has been used to characterize ,and trace,
the phylogeny of diverse plant and animal species.

24. Cell /tissue of an individual
Double stranded genomic DNA
Single stranded DNA
Primers annealed to template DNA
Complementary strand is synthesized
Isolate DNA
-Taq polymerases ,primer, dNTPs
- Denature DNA @ 94 c° for 1 minute
- keep in tubes of PCR thermocycler
Annealing of primers ( 36° c for 2 min)
DNA is synthesized at 72° c for 1.5 min
Separate amplified products by gene electrophoresis
35 – 45 cycles

25. •Quick and easy to assay.
•Low quantities of template DNA required.
•Dominant markers.
•In expensive.
•Do not require any specific knowledge of the target.
• No species specific probes are required for different species .
• Crude DNA preparation may be used for analysis of whole genome
• It does not require blotting or hybridization

26. •Low reproducibility
•Highly sensitive and complicated procedure.
•PCR cycling conditions greatly influence the out come.
•Mismatches between primer and template may result in total absence of
PCR product.
• RAPD markers are dominant so it is cause problems whether the DNA
segment is amplified from locus that heterozygous ,homozygous.

27. •Gene mapping
•DNA amplification finger printing
•Study of closely related species
• RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-
PCR).
• Certain genetic markers may be tagged at specific regions in the genome

28. •The term microsatellites was coined by Litt & Lutty (1989)and it also known as Simple
Sequence Repeats (SSRs), are sections of DNA.
•Microsatellite markers, developed from genomic libraries, can belong to either the
transcribed region or the non transcribed region of the genome.
• Microsatellite sequences are especially suited to distinguish closely related genotypes;
because of their high degree of variability, they are, therefore, favoured in population
studies .
• Microsatellite polymorphism can be detected by Southern hybridization or PCR.
•If nucleotide sequences in the flanking regions of the microsatellite are known, specific
primers can be designed to amplify the microsatellite by PCR.
•microsatellite may be identified by screening sequence databases,
polymorphism can detected by gel electrophoresis
SSR (SIMPLE SEQUENCE REPEAT) OR MICROSATELLITES

29. Microsatellites – Properties
 Co-dominant
 Inherit in Mendelian Fashion
 Polymorphic loci with allele number as high as 14 – 15 per
locus
 Mostly reported from non-coding region, hence can be
independent of selection
 Flanking region is highly conserved in related species
 Can be obtained from small amounts of tissues [STR
analysis can be done on less than one billionth of a gram
(a nanogram) of DNA (as in a single flake of dandruff)]
 PAGE separation; silver staining/automated genotyping
 Abundant in the eukaryote genome (~103 to 105 loci
dispersed at 7 to 10100 kilobase pair (kb) intervals)

30.  Also called as STR, SSR.
 Tandemly repeated DNA sequences with the repeat/size
of 1 – 6 bases repeated several times
 Highly polymorphic; can be analyzed with the help of
PCR
 Individual alleles at a locus differ in number of tandem
repeats of unit sequence owing to gain of loss of one or
more repeats and they can be differentiated by
electrophoresis according to their size
 Powerful DNA markers for quantifying genetic variations
within & between populations of a species

31. Short Tandem Repeats (STRs)
the repeat region is variable between samples while the
flanking regions where PCR primers bind are constant
7 repeats
8 repeats
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved
from one another

32. •The microsatellite, or short sequence repeat (SSR), is
a powerful genetic marker, useful in many areas of
fish genetics and breeding.
•Polymorphic microsatellite loci have been frequently
applied to the analysis of genetic diversity, population
genetic structure, and genomic mapping.
•These co-dominant markers have also been applied
to the classification and systematics, parentage
identification, germplasm conservation, and breeding
programme of food fish.

33. • Because the technique is PCR-based, only low quantities of template DNA
(10–100 mg per reaction) are required.
•The strengths of microsatellites include the co-dominance of alleles, their
high genomic abundance
• the reproducibility of microsatellites is high and analyses do not require high
quality DNA

34. • Main drawbacks of microsatellites is that high development costs
• Errors in genotype scoring
•Difficulty in interpretation
• Previous genetic information is needed
• Huge upfront work required
• Problems associated with PCR of microsatellites

36. FORENSICS
•Microsatellite loci are widely used for forensic identification and relatedness testing and
are predominant genetic marker in this area of application
•In forensic identification cases ,the goal is typically to link a suspect with a sample of
blood semen, or hair taken from a crime
•Another application involves linking DNA samples with relative of a missing person,
because the length of microsatellite may vary from one person to next .(finger printing)
DIAGNOSIS AND IDENTIFICATION OF HUMAN DISEASES
• Microsatellites changes in length early in the development of some cancers , they are
useful markers for early cancer detection
• It is useful in linkage studies so it help to locate genes responsible for various genetic
disorders

37. POPULATION STUDIES
• By looking at the variation of microsatellites in populations,
inferences can be made about population structures and differences ,
genetic drift, genetic bottlenecks and even date of a last common
ancestor.
CONSERVATION BIOLOGY
• Used to detect sudden changes in population
• Effect of population fragmentation
• Interaction of different population
• useful in identification of new and incipient populations

38. MINISATELLITE DNAs
•These are usually 0.2 to 2 kb long and consists of tandem repeats of 9
to 40 bp sequences
• In the case of humans ,minisatellite DNAs are concentrated in the
terminal regions of the chromosomes
• Therefore they do not contribute a good marker system for human
genome ,despite the great degree of polymorphism exhibited by them
• They are useful in the finger printing

39. VNTR ( VARIABLE NUMBER OF TANDEM REPEATS)
•Repetitive DNA composed of copies of short sequences, involved in generation of
polymorphic loci that are useful in genetic fingerprinting , also known as hyper variable
regions.
• A variable number of tandem repeat is a location in a genome where a short
nucleotide sequence is organized as a tandem repeat.
• Tandem repeats occur in DNA when a pattern of 2 or more nucleotide is repeated and
the repetitions are directly adjacent to each other.
• e.g.; A-T-T-C-G-A-T-T-C-G-A-T-T-C-G In which the sequence A-T-T-C-G is repeated 3
times
• These can be found on many chromosomes ,and often show variations in length
between individuals. each variant act as an inherited allele, allowing them to be used
for personal or parental identification
•Their analysis is useful in genetics and biology research , forensic, and DNA finger
printing

40. • VNTRs are dispersed throughout the genome and are made up of a variable number of
end to end duplications of identical or almost identical sequences of 12 -80 bp each
• polymorphism in VNTRs is usually associated with the number of repeats.
• These variations constitute the allele of VNTR loci, polymorphism also arises due to a
variation in the distribution pattern of repeated sequences in the different alleles.
• VNTRs were in important source of RFLP genetic markers used in linkage analysis of
genome
• VNTRs have become essential to forensic crime investigation, via fingerprinting
•When removed from surrounding DNA by PCR or RFLP methods, and their size determined
by gel electrophoresis or southern blotting ,they produce a pattern of bands unique to
each individual.
• VNTRs analysis is also being used to study genetic diversity and breeding patterns in
populations of wild or domesticated animals

41. SNPs ( single nucleotide polymorphism)
GCTGTATGACTAGAAGATCG
AT
GCTGTATGACGAGAAGATCG
AT•IT IS VARIATION IN A SINGLE NUCLEOTIDE WHICH MAY OCCUR AT SOME SPECIFIC POSITION
IN THE GENOME WHERE EACH VARIATION IS PRESENT TO SOME APPRECIABLE DEGREE
WITH IN A POPULATION.
OR
•A SNP IS DEFINED AS A SINGLE BASE CHANGE IN A DNA SEQUENCE THAT OCCURS IN A
SIGNIFICANT PROPORTION (MORE THAN 1 PERCENT) OF A LARGE POPULATION.

42. •In human beings, 99.9 percent bases are same.
•Remaining 0.1 percent makes a person unique.
•Different attributes / characteristics / traits
•how a person looks,
•diseases he or she develops.
•These variations can be:
•Harmless (change in phenotype)
•Harmful (diabetes, cancer, heart disease, Huntington's disease,
and hemophilia )
•Latent (variations found in coding and regulatory regions, are not
harmful on their own, and the change in each gene only becomes
apparent under certain conditions e.g. susceptibility to lung
cancer)

43. ►SNPs are found in
coding and (mostly) noncoding regions.
►Occur with a very high frequency
about 1 in 1000 bases to 1 in 100 to 300 bases.
►The abundance of SNPs and the ease with which they can be
measured make these genetic variations significant.
►SNPs close to particular gene acts as a marker for that gene.
►SNPs in coding regions may alter the protein structure made by
that coding region.
SNP FACTS

44. •Single-nucleotide polymorphisms may fall within coding sequences of genes, non-
coding regions of genes, or in the intergenic regions (regions between genes).
•SNPs within a coding sequence do not necessarily change the amino acid sequence
of the protein that is produced, due to degeneracy of the genetic code.
•SNPs in the coding region are of two types,
• synonymous
• nonsynonymous
• Synonymous SNPs do not affect the protein sequence
•nonsynonymous change the amino acid sequence of protein. The nonsynonymous
SNPs are of two types: missense and nonsense.

45.  IN DISEASE DIAGNOSIS
 IN FINDING PREDISPOSITION TO DISEASES
 IN DRUG DISCOVERY & DEVELOPMENT
 IN DRUG RESPONSES
 INVESTIGATION OF MIGRATION PATTERNS
ALL THESE ASPECT WILL HELP TO LOOK FOR MEDICATION &
DIAGNOSIS AT INDIVIDUAL LEVEL
SIGNIFICANCE OF SNPs

46. 1. SNPs ARE THE MOST FREQUENT FORM OF DNA
VARIATIONS
2. THEY ARE THE DISEASE CAUSING MUTATIONS IN
MANY GENES
3. THEY ARE ABUNDANT & HAVE SLOW MUTATION RATES
4. EASY TO SCORE
5. MAY WORK AS THE NEXT GENERATION OF GENETIC
MARKERS

47. •SNPs are used for identification and forensics
•SNPs are used for mapping and genome-wide
association studies of complex diseases
•SNPs are used for estimating predisposition to
disease
•SNPs are used for immigration & citizenship in the UK
•SNPs are used to predict specific genetic traits
•SNPs are used for classifying patients in clinical trials