DNA AMPLIFICATION MARKERS -RAPD -SSR/ISSR -FISH-DNA PRESENTED BY- SINCHANA A.R

Genetic markers Genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed. Types of markers: RAPD,SCAR,SSR,RFLP,SSLP, AFLP etc.

RAPD RAPD stands for 'Random Amplified Polymorphic DNA'. It is a type of PCR reaction, but the segments of DNA that are amplified are random. It was discovered by williams and is a commonly used genetic diversity marker. RAPD markers are decamer (10 nucleotide length) are the DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to distinguish between genetically distinct inidividuals.

PRINCIPLE The principle is that, a single, short oli-gonucleotide primer, which binds to many different loci, is used to amplify random squences from a complex DNA template. This means that the amplified fragment generated by PCR depends on the length and size of both the primer and the target genome. The assumption is made that a given DNA sequence (complementary to that of the primer) will occur in the genome, on opposite DNA strands, in opposite orientation within a distance that is readily amplifiable by PCR

The use of a single decamer oligonucleotide promotes the generation of several discrete DNA products and these are considered to originate from different genetic loci. Polymorphisms result from mutations or rearrangements either at or between the primer binding sites and are detected as the presence or absence of a particular RAPD band. No fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

RAPD reaction 1:

RAPD reaction 2:

Reaction 1: The arrows represent multiple copies of a primer (all primers (arrows) have the same sequence). The direction of the arrow also indicates the direction in which DNA synthesis will occur. The numbers represent locations on the DNA template to which the primers anneal. Primers anneal to sites 1, 2, and 3 on the bottom strand of the DNA template and primers anneal to sites 4, 5, and 6 on the top strand of the DNA template. Product A is produced between primers 2 &5 and B between 3&6. Product is not produced between 1&4 (too apart) and between 2&2/3&5 (wrong orientation)

Finding difference between genomes using RAPD reaction 2: Suppose there was a change in sequence at primer annealing site #2: As shown in this figure, the primer is no longer able to anneal to site #2, and thus the PCR product A is not produced. Only product B is produced. If you were to run the 2 RAPD PCR reactions diagramed above on an agarose gel, there is formations of bands. (RAPD bands)

Advantages : -high number of fragments. -it requires only small amount of DNA and the procedure can be automated. -it involves no blotting or hybridization steps.(so.quick simple and efficient) - No prior knoweledge of the genome sequence used is required.

Disadvantages: -Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). - Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

SSR Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are repeating sequences of 2-5 base pairs of DNA. Microsatellites are typically co-dominant. It is 1-6 nucleotides long. They are present more in number in the arm of the chromosome. In humans 5 most abudant microsattelites contain repeat sequence of A, AC, AAN, AAN, AG (N=C,G,T)

TYPES: Perfect repeats – CACACACACACACACA Imperfect repeats- CACACA-------CACACA------- Mono repeats (based on number of bp)- CCC/AAA Di repeates – CACACACA Tri repeats – CCA CCA CCA Tetra repeats – GATA GATA GATA

The region containing the microsatellite is amplified by PCR using primers that flank the microsatellite. The amplified DNA is then run out on a gel that will separate DNA fragments based on size. Individuals typically have two alleles for all microsatellites (the reasons why they would have one are discussed below). If the number of repeats of one allele is different from the other, then two separate bands would show up on the gel.

Here individual alleles at a locus differ in no Here individual alleles at a locus differ in no.of tandem repeats of unit sequence owing to gain or loss or one or more repeats , and they can be differentiated by electrophoresis based on their size. The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and that base pair on either side of the repeated region. Therefore a single pair of PCR primers will work for every individual in the species and produce different sized products for different length of microsatellites.

properties -They are co-dominant. -They are mostly found in non coding regions (introns). So the changes in the number or length will not affect the gene function. -They are abundant in eukaryotic genome. -Inherit in Mendelian fashion. -Flanking region is highly conserved in related species.

ISSR Inter simple sequence repeat. The complementary sequences to two neighboring microsatellites are used as PCR primers; the variable region between them gets amplified. Used in DNA finger printing, population studies, genetic mapping, polymorphism studies etc It is not useful in distinguishing individuals. It is a dominant marker.

Principle

FISH DNA Fluorescent in situ hybridization is a technique which is used to detect and localise the presence or absence of specific DNA sequence on chromosomes. It uses fluorescent probes which bind only to those parts of chromosome with which they show a high degree of sequence similarity. It is highly useful in detecting chromosomal abnormalities.

PRINCIPLE The sample DNA from metaphase chromosome or interphase nuclei is first denatured where the complementary strands are seperated within the DNA helix. The fluorescently labelled probes is then added to the denatured mixture and it hybridizes with the sample DNA at target site and reanneals back to the double helix. The probe signal can then be seen through a fluorescent microscope and sample DNA is scored for the presence or absence of the signal.

FISH can be used in metaphase cells to detect specific microdeletions which is otherwise not possible through routine cytogenetics . It is also used to identify extra material of unknown origin, chromosome rearrangements (in case of cancer cells). Microdeletion syndrome currently diagnosable with FISH are : cri-du-chat , steroid sulfatase deficiency , smith-magenis syndrome etc

FISH can be used in interphase cells to detect rearrangements that are characteristic for certain cancer s. The primary advantage of interphase FISH is that it can be performed very rapidly within 24hrs because cell growth is not required. Fibre FISH is a new version which is used for cytogenetic studies.

APPLICATIONS It can be used to map sequence to a specific position on chromosome. It can also be used to do chromosome painting to make a comparision between two species by using DNA from entire chromosome or even from entire genome of one of the species as a probe on the other. It is also used in field of microbial ecology and clinical studies ( to detect if a patient is infected with particular pathigen or not )

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