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Sujit kumarFB-MA2_01

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INTRODUCTION

Molecular markers- ?

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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

iat

Evolution Of Molecular Markers

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Subjective view of the changing relative importance of

different molecular markers(Schlotterer C, 2004)

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• What is isozyme?

•

What is allozyme?

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Principle

enzymes proteinAmino

acidDNA

Isozyme- variation in size, charge and some time intertiary confirmation

Charge variation - due to substitution of basicamino acid by acidic amino acid

Only large variation - detected by electrophoresis

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METHODOLOGY

Sample Selection And Preparation

Electrophoresis

StainingGenetic Interpretation

Genetic Interpretation

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• 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

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Continue………………

• Chelating agents, protease inhibitors, and

enzyme stabilizers, such as EDTA,

polyvinylpyrrolidone, PMSF, and dithiothreitol,

can be added to the sample buffer to increaseresolution.

•

The addition of small quantities of substrate (20mg/100 ml sample buffer) may also help to

stabilize some enzymes.

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Electrophoresis

• Traditionally, isozyme analysis was

performed with starch or PAGE, but

isoelectric focusing is now being usedmore commonly.

•

SDS-PAGE - cannot be used for isozymeanalysis.

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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.

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GENETIC INTERPRETATION

• Homozygous or Heterozygous

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• 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.

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• 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

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Number of loci coding for enzyme

• May be by one or more loci

• For example, haemoglobin is a tetramer with

two α subunits and two β subunits (where the

α and β subunits are coded by different

genes).

• On gel it is possible to differentiate between

the loci

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The reason of development of different band is tht

isozyme of enzyme may present in differentcellular compartment

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• But in certain case where , even after long run

of gel loci are not differentiable , in such case

enzyme is called as unresolvable .

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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

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Eight individuals of a diploid plant species in

which samples have been stained for a

hypothetical enzyme (XYZ):

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Application

• Population genetics studies

• Rates, (sub) population structure and populationdivergence.

•

Allozymes are particularly useful at the level of conspecific (same species) populations and closelyrelated species- so useful to study diversity

• They have been used, often in concert with othermarkers in diversity studies, to study interspecificrelationships, the mode of genetic inheritance, andallelic frequencies in germplasm collections and toidentify parents in hybrids.

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Comparison between Allozyme marker

and DNA based marker

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