Cytogenetic RFLP (Restriction Fragment Length Polymorphism) Maps Using PCR ( Polymerase Chain Reaction)

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	Cytogenetic RFLP (Restriction Fragment Length Polymorphism) Maps Using PCR ( Polymerase Chain Reaction) - Assignment of protein encoding genes to specific chromosomes is a major goal of human cytogenetics. This can be routinely achieved by hybridizing a cloned gene, with Southern blots or dot blots prepared from DNA samples extracted from hybrid cell lines of known cytogenetic composition. The Southern blots in this case allow rapid identification of a chromosome specific restriction fragment of the gene of interest. However, in this exercise, pseudogenes and genes related with the probe or sequences in highly conserved gene may give a false positive signal, so that not all restriction fragments so identified may represent the gene of interest.
Therefore a gene may not be assigned to a specific chromosome with complete certainty. In view of these difficulties in chromosome mapping, alternative approach was recently utilized to map the gene p68 in humans. For this gene, the conserved region was already known and for the synthesis of primers to be used in PCR amplification. Species specific differences could be detected in PCR products, the difference 5bp difference) being due to intron lengths. For p68 genes, it was shown at PCR products using two specific primers differed in mouse and humans in band position. This property was used in screening a number f hybrid cell line of known cytogenetic composition and the results from number of cell lines could be compared to find out the chromosome. In which the gene may be located.
The hybrid cell line PCTBA1.8 contained only one human chromosome i.e. human chromosome 17 and INA from this line gave a human specific restriction fragment in PCR product thus confirming that p68 gene was present on chromosome 17 later, hybrid cell lines having either only one or more fragments of human chromosome 17 were derived. The use of these cell lines enabled the happing of p68 gene to the distal part of the long arm of chromosome in the region between the bands 17q23 and 17q25.