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Today ’ s Lecture Genetic mapping studies: two approaches Classical linkage map/genome-wide association study Physical map Cloning and isolating genes the old-fashioned way using positional cloning Search for the cystic fibrosis gene Next Lecture Modern genome sequencing
Genetic linkage mapping involves determining the statistical association of specific traits with genetic markers on chromosomes using pedigrees and crosses.
5,264 microsatellites to 2,335 chromosome loci
(average density of one marker every 599 kb)
High-density genetic map of 5,264 microsatellites localized to each of 23 chromosomes.
Physical map = map of physically identifiable regions of genomic DNA constructed without recombination analysis.
1. Low Resolution-Cytogenetic/FISH maps
“q” = long arm
“p” = short arm
Numbered from the centromere starting with “1”
2. High Resolution-YAC/BAC Clone Contig Maps
Fig. 10.1 2nd edition, YAC contig physical map assembled by microsatellite mapping (combination YACs + microsatellite mapping)
e.g., cloning and discovery of the cystic fibrosis (CF) gene.
CF results from defect in protein that regulates the movement of salt and water in and out of cells.
Causes thick mucus secretions in the lungs, pancreas, and intestines.
Causes lung disease and organ failure, patients experience chronic bacterial infections.
Life expectancy is abut 40 years.
Many hundreds of individuals with CF pedigrees were screened with a large number of RFLPs.
A single recurring RFLP showed weak linkage (statistical association) to the cystic fibrosis trait.
CF gene was next localized to chromosome 7 using a labeled RFLP probe and in situ hybridization to condensed chromosomes.
All other known RFLPs from chromosome 7 were simultaneously screened for linkage to CF.
Two more linked RFLPs were discovered on a 500,000 bp subregion (31-32) of the long arm of chromosome 7 (7q31-q32).
The data indicated CF locus is within a 500,000 bp region of chromosome 7.
chromosome walking was used to
identify a candidate gene for a disease like cystic fibrosis.
Use partial restriction digestion to cut a large section of chromosomal DNA into large overlapping fragments.
Circularize fragments with DNA ligase, bringing ends of DNAs thatpreviously were distant close together.
Cut the circles with a restriction enzyme yet again to release the junction region (ends are now inverted).
Clone junction regions to form a jumping library.
Subclone a small fragment of DNA and use as a probe to find the next junction fragment occurring in the library (same technique as chromosome walking).
Repeat… and/or start chromosome walking.
Chromosome jumping reaches the target gene faster than walking.
Similar technique called “mate pair” is used in today’s next-generation sequencing.
*Genes are more conserved than non-coding sequences and similar sequences should be found in other species.
cDNA (mature mRNA of same size) is 6,500 bp.
Genomic DNA: CF gene spans 250 kb and contains 24 exons.
68% of Caucasians with cystic fibrosis show a 3-bp deletion that results in the loss of phenylalanine (Phe).
Sixty other mutations described.
Fig. 4.13, CFTR Structure