Restriction Fragment Length Polymorphisms Rflps, Homozygotes and Heterozygotes, Obtained Digestion, Restriction Enzymes, Molecular Probe Hybridization

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Restriction fragment length polymorphisms (RFLPs).

RFLPs are based on polymorphisms

arising due to base substitutions, insertions, deletions and translocations that might have occurred in the past in specific regions of genomic DNA.

These changes lead to differences in the size of restriction fragments obtained due to digestions with specific only in a fraction of these fragments, that are related with other homology with a molecular probe used for hybridisation .

The principle involved in the detection of RFLPs is illustrated in and 9.2. Following steps are involved:

(i) digestion of genomic DNA with individual restriction enzymes, followed by electrophoresis on agarose gel (1 %-2%);
(ii) transfer of DNA fragments from the gel to a filter following the technique of Southern blotting

The Technique Involved in the Detection of Restriction	The Results of Digestion of Genomic DNAs of Four Hypothetical Individual with Three Enzymes

(iii) hybridization of DNA fragments on the filter with one or more molecular probes individually (the probes need to be labelled radioactively with 32P or non-radioactively with biotin/digoxigenin, before hybridization), and
(iv) study of polymorphism in the hybridization patterns.
The molecular probes used for RFLP can be either genomic probes (derived from genomic DNA) or cDNA probes (derived from mRNA), which can be developed from genomic/cDNA libraries in the laboratory or else can be procured from other laboratories

Heterologous probes derived from one species can also be used across related species and genera. Rarely, synthetic oligonucleotides may also be used as molecular probes.

The level of polymorphism detected through RFLPs depends both on the restriction enzyme used for digestion and the probe used for hybridization.

One of the advantages of RFLPs is due to their co-dominant nature, which allows distinction between homozygotes and heterozygotes

(co-dominance means that, fragments of different sizes available in two parents are allelic and both can be visualised in the F1 hybrid).

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Another advantage of RFLPs is due to the known homology between the polymorphic fragments scored, since they hybridize with the same probe (compare with RAPDs).

RFLP analysis, however, is time consuming, labour intensive and too slow for rapid evaluation of large segregating populations used in a commercial breeding programme.

An addition disadvantage for RFLP analysis is due to large quantities of genomic DNA (10µg or more) needed for the preparation of Southern blots.

Because of these limitations, and due to superiority and cost-effective nature of PCR based marker systems, which became available, later, RFLPs are seldom, if ever used now for developing markers for a variety of purposes

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Amplifed Fragment Length Polymorphism (AFLP) Single Strand Conformation Polymorphism (SSCP) Molecular Markers based on PCR Followed by Hybridization Oligonucleotide Fingerprinting Using RAPD/MP-PCR Framents Sequence based Molecular Markers Single Nucleotide Polymorphisms (SNPs) Gel-based Assays of PCR Products for SNP Detection Non-Gel based Assays of PCR Products for SNP Detection Taqman Assay Use of Molecular Beacon Oligonucleotide Ligation Assay (OLA) DNA Chip/Microarray Dynamic Allele-specific Hybridization Minisequencing Pyrosequencing for SNP Genotyping Temperature Modulated Heteroduplex Analysis TMHA Using DHPLC Wave^TM System A Non PCR Invasive Clevage Assay and Maldi TOF-MS for SNP Detection Comparison of Different Types of Molecular Markers High Throughput Approach in Molecular Marker Technology Comparison of General Features of Different types of Molecular and their use