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Sujit kumar FB-MA2_01

INTRODUCTION Molecular markers- ?

iat Evolution Of Molecular Markers

Enzyme based markers e.g. Allozymes

DNA based markers e.g. RFLPs, Minisatellit es

PCR based markers e.g. Microsatellit es, RAPDs, ISSRs, IRAPs, AFLPs

Subjective view of the changing relative importance of different molecular markers


(Schlotterer C, 2004)

What is isozyme? What is allozyme?

Principle
enzymes protein Amino acid DNA

Isozyme- variation in size, charge and some time in tertiary confirmation Charge variation - due to substitution of basic amino acid by acidic amino acid
Only large variation - detected by electrophoresis

METHODOLOGY
Sample Selection And Preparation

Electrophoresis

Staining Genetic Interpretation

Genetic Interpretation

Most critical step The number of isolates and their geographic and host range will all affect data interpretation. Denaturation of enzyme must be prevented during and after sample preparation and storage Maintain temperature below 4 C during collection and storage Repeated freezing and thawing denature the enzyme

Continue
Chelating agents, protease inhibitors, and enzyme stabilizers, such as EDTA, polyvinylpyrrolidone, PMSF, and dithiothreitol, can be added to the sample buffer to increase resolution. The addition of small quantities of substrate (20 mg/100 ml sample buffer) may also help to stabilize some enzymes.

Electrophoresis
Traditionally, isozyme analysis was performed with starch or PAGE, but isoelectric focusing is now being used more commonly.
SDS-PAGE - cannot be used for isozyme analysis.

Staining
Staining solution with enzyme specific substrate and cofactor Enzymatic reaction gives colored product Fluorescent products - detected with ultraviolet light. Non fluorescent products - visualized as negatively stained by reacting the starch with a fluorescent compound.

GENETIC INTERPRETATION
Homozygous or Heterozygous

Enzyme may be monomer, dimer, trimer or tetramer depending upon number of subunit involved as shown below

This particular aspect of enzyme structure is responsible for the appearance of additional bands in the heterozygotes.

Total number of bands in a heterozygote should consist of one more than the number of subunits in the enzyme ( five bands in a tetramer). Consider the following dimeric enzyme

Number of loci coding for enzyme


May be by one or more loci For example, haemoglobin is a tetramer with two subunits and two subunits (where the and subunits are coded by different genes). On gel it is possible to differentiate between the loci

The reason of development of different band is tht isozyme of enzyme may present in different cellular compartment

But in certain case where , even after long run of gel loci are not differentiable , in such case enzyme is called as unresolvable .

Summary of Genetic Interpretation


There are four important aspects that should be known about any particular isozyme system before the gels can be scored correctly: 1. Structural configuration - monomer, dimer, etc. 2. Number of loci involved 3.Cellular location - cytosol, plastid, mitochondria, 4. Ploidy level of the species

Eight individuals of a diploid plant species in which samples have been stained for a hypothetical enzyme (XYZ):

Application
Population genetics studies Rates, (sub) population structure and population divergence. Allozymes are particularly useful at the level of conspecific (same species) populations and closely related species- so useful to study diversity They have been used, often in concert with other markers in diversity studies, to study interspecific relationships, the mode of genetic inheritance, and allelic frequencies in germplasm collections and to identify parents in hybrids.

Comparison between Allozyme marker and DNA based marker