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Home >>Molecular Maps of Animal Genome >>Positioning of a Cloned Sequence on the Chromosome
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Positioning of a Cloned Sequence on the Chromosome - The position on chromosome can also be located using the, following steps:

(i) The two linear molecules are designed as YCF3 and YCF4 vectors. Since the centromere telomere orientation of the segment is not known, four molecules must be constructed with both orientations of segment inserted into each vector.

This can be done by cutting the vector at a specific site in polylinker and introducing the segment with compatible ends. The vector carrying the segment is linearized, leaving the telomere at one end and the DNA segment at the other;

(ii) The four linear molecules are constructed and used to transform the strain YNN290, having useful markers.

(iii) Transformants are streaked onto rich media plates and grown for three days to allow the colony colour to develop.
(iv) Intact chromosome DNA is prepared from 5-10 pink colonies for each, YCF3 and YCF4.
(v) DNA is separated by alternating CHEF gel electrophoresis and transferred to nylon membrane.
(vi) The blots are hybridized with mapping sequence.
(vii) Two bands were identified to represent two chromosome segments and were sized using phage DNA fragments or the chromosomes of YPH149 (a strain of yeast).
(viii) The mapping sequence is positioned and confirmed by rehybridizing the membrane with one or more probes earlier mapped.
The advantage of the present method also lies in the fact that a cloned sequence can be mapped without preparing detailed restriction map as done in chromosome walking.

 

  

 
 

 

 
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