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Home >>Molecular Maps of Animal Genome >>Molecular Markers - Restriction Fragment Length Polymorphism - RFLPs
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Molecular Markers - Restriction Fragment Length Polymorphism - RFLPs - RFLPs as molecular markers, and the procedure utilized for their detection, while discussing the uses of molecular probes. In eukaryotes, the repeated sequences being common, often only unique DNA sequences are used as RFLP probes.

These DNA sequences may be obtained either as cDNA (complementary DNA) derived from mRNA (extracted from any metabolically active tissue) or as genomic DNA. For the purpose of preparing RFLP maps, generally probes are prepared from genomic DNA using the following steps:

(i) DNA is extracted from cultured cells, or ,tissues;

(ii) DNA is digested with a restriction enzyme like Pst I, which is methyl sensitive, so that repetitive sequences are not cleaved (repetitive sequences are heavily methylated);

(iii) digested DNA is fractionated using a sucrose gradient (10%-40%) and DNA fragments in the size range of 500-4000bp are separated;

(iv) the size fractionated fragments are cloned in a plasmid (pUC18 or pBluescript), and used for transforming efficient E. coli cells;

(v) transformed bacteria are plated and those with inserts (DNA cloned in plasmid) are selected through white/blue colour of colonies (white colonies indicate presence of insert. due to disruption of lac Z gene; blue colonies have no inserts);

(vi) selected bacterial colonies are rechecked for the presence of the inserts by cleaving the plasmids and subjecting them to electrophoresis;

(vii) the selected clones may be checked, if they all represent unique sequences; this can be achieved by hybridizing these clones with labelled genomic DNA using colony hybridization or dot blots, so that only those clones will represent unique sequences, which show weak signal or no signal;

(viii) the selected clones maybe radioactively labelled and used for hybridization of Southern blots derived from genomic DNA digested with a restriction enzyme like BamHI; only those clones are now selected, which give only one or two bands on autoradiograms, and those giving many bands are rejected;

(ix) from these selected clones, one can select those clones, which give polymorphism in the parents used for RFLP mapping.

 
 

 

 
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