MOLECULAR MARKERS

•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…

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.

MOLECULAR MARKERS

Based on mode of transmission & evolutionary dynamics genetic Markers

can be categorized in to

Protein markers such as alloenzymes

DNA markers such as: MITOCHONDRIAL DNA MARKERS

NUCLEAR DNA MARKERS ( RAPD , VNTR SUCH AS MICRO & MINI SATELLITES)

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.

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

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.

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

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 separatelycleaved by restriction enzymes ,passed through electrophoresis.

Then it blotted on nitrocellulose membrane and probed with a radio labelledDNA.

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.

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

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

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

•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

•Require large quantities of high molecular weight DNA.

•Expensive process

•Time consuming

•Labor intensive

DISADVANTAGES

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

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)

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

• Need for purified, high molecular weight DNA.

• The major disadvantage of AFLP markers is that these are dominant markers.

•Abundance of data.

DISADVANTAGES

•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

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.

•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.

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

•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

ADVANTAGES

•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.

DISADVANTAGES

•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

APPLICATIONS

•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

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)

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

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

•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.

• 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

ADVANTAGES

• 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

DISADVANTAGES

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

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

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

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

• 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

SNPs ( single nucleotide polymorphism)

GCTGTATGACTAGAAGATCGATGCTGTATGACGAGAAGATCGAT•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 DEGREEWITH 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.

•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)

SOME FACTS

►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

•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.

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

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

ADVANTAGES

•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

APPLICATIONS