Module 7: Molecular Cell Biology

Front 1

Positional Cloning
DMD
CF

Back 1

-We can clone a gene by forming a self-replicating DNA vector. Restriction enzymes are used to cut out the DNA of the gene of interest. The same restriction enzyme is used to create a linear DNA molecule from a purified plasmid. We then insert the DNA into the plasmid where the two will combine and anneal using their sticky ends and DNA ligase. We can then transfect a bacterial cell with the plasmid where it will continue to self-replicate.
-Using a variation of the plus-minus screening technique, they were able to isolate several clones that were deleted in the patient. One of them would prove to contain the DMD gene (chromsome walking + southern blots)
-linked to chromosome 7 by linkage analysis using RFLPs. By looking for more tightly linked RFLPs, scientists showed that the gene is contained within an ~1.5Mb region (chromosome walking + jumping)

Front 2

Linkage map

Back 2

Linkage maps use RFLP's and related genetic markers to enable researchers to localize genes with pedigree analysis

Front 3

Physical map

Back 3

In order to then clone a localized gene, a physical map is necessary. The procedure used in model organisms, such as the nematode and yeast, is to screen a genomic library with large inserts (usually in YACs) to find, and order, a series of overlapping clones, called contigs.

Front 4

Human Genome Project

Back 4

-able to describe those genes that are involved in diseases.
-bring early detection and treatment of disease, and perhaps prevention.
-help speed the task to find the genes that cause these diseases