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Use of Flow Sorted and Microdissected Chromosomes -In mouse and human, individual chromosomes can be separated from mouse human hybrid cell lines using the technique of now sorting. Both normal and translocated human chromosomes could be obtained through flow sorting.

In mouse, the chromosomes being similar in size, only five peaks were obtained in flow sorting and individual chromosomes could not be easily separated except when all chromosomes except chromosomes 19 and X were fused in a metacentric arrangement. This technique allowed the construction of chromosome specific-libraries in A (lambda) vector.

X­ chromosome specific cDNA clones 'were also identified bl2 probing A-phage libraries prepared from flow sorted X chromosome with 2p labeled cDNA probes derived from hamster human cell hybrid cell lines containing the X chromosome as the only human chromosome.

Only X-specific cDNA clones will hybridize, which will represent the transcribed sequences on X- chromosome and will thus allow the construction of a map of X-chromosome, involving functional genes only. A phage library was also prepared using flow sorted chromosome material enriched for chromosome 16.

In recent years, micromanipulators have also been used for isolating specific chromosomes (where specific chromosomes can be identified), both in animals and plants. Once these specific individual chromosomes are separated in large number they can be used for microcloning to give chromosome specific clones.

Fragments of chromosomes representing specific regions, can also be dissected physically from metaphase spreads of chromosomes and used for microcloning. This will give chromosome region specific (or band specific) libraries to be used eventually for avariety of purposes including the physical mapping.

In Drosophila, polytene chromosomes have been used for microdissection and microcloning, leading to the preparation of physical maps. Techniques have also been developed for the precise dissection of single bands from human chromosomes, using siliconized glass needles and electronically controlled micromanipulator.
The DNA from these chromosome fragments is then extracted, digested with an enzyme, cloned, and amplified using polymerase reaction (PCR) to give band specific DNA libraries. As few as 20 detected chromosome fragments are enough to give such a library.
Since the dissected chromosome fragments will not give enough quantity of DNA on extraction, PCR technology for amplification has particularly proved very useful in this connection.
These chromosome sorting and microdissection techniques can be easily applied to construction of chromosome specific or band specific genomic libraries, leading to the construction of chromosome maps

 
 

 

 
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