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