Study Guides (238,207)
Canada (115,009)
Biology (1,419)

Genetics Lecture No. 10.docx

3 Pages
Unlock Document

Western University
Biology 2581B
Jim Karagiannis

Genetics Lecture No. 10: Mapping & Sequencing th Monday February 11 , 2013 Partitional Cloning: -Positional cloning involves finding the location of a gene based on its phenotype (a specific location for a specific gene) and can be done with (as in the cystic fibrosis example) and without (as in the hemophilia A example) mapping information. In order to clone a gene without mapping information, we need information about family history and an idea of what might be causing the mutation and what its protein might be (biological as well as genetic information). Hemophilia A Cloning & The Blood Clotting Cascade: -In this positional cloning example, we wish to find the gene causing hemophilia A, where patients of this disease fail to clot their blood (wounds keep on bleeding). In hemophilia A patients, their disease is caused by an X-linked recessive mutation (more prevalent in males due to female carriers) on a single gene which encodes a defective protein (known as coagulation factor VIII) that fails to associate fibrin into a cross-linked network (necessary for forming a blood clot). - In this same blood clotting cascade, coagulation factor II (aka prothrombin) is cleaved to become thrombin (a serine protease), which then converts fibrinogen (soluble glycoprotein that doesn’t clot) to fibrin (insoluble fibre which does clot through cross-linkage). Blood tests can determine whether an active form of each factor involved in the clotting cascade is present. The results of such analyses show that many hemophiliacs, lack an active factor VIII in their blood. Researchers purified factor VIII, determined its amino acid sequence, used this information to infer all possible degenerate coding sequences (recall wobble position), constructed oligonucleotides for a region with minimal degeneracy, probed a genomic library with these oligonucleotides, and obtained genomic clones of the F8 gene. Colony Hybridization & The Positional Cloning Of Hemophilia A: -By growing E. coli colonies containing different DNA vectors from a genomic library, we can then add the degenerate probe which is part of the hemophilia version of the factor VIII. We then observe if any of the E. coli have a vector complementary to the VIII sequence and find that only one colony is containing a single vector. We then isolate the plasmid DNA from that single colony and sequence the DNA. The results demonstrated a quite complex gene whose mutated allele causes hemophilia A. In order to confirm that the correct gene was sequenced from patients having hemophilia A, it is necessary to make primers for people who do not have hemophilia and those who do (mutation should be in patients, but not in normal people). The results showed that there are different types of mutations identified causing hemophilia in that gene: base substitutions (between important amino acids are sufficient for causing the disease), splice mutations, small/large deletions. Cystic Fibrosis & Positional Cloning: -Cystic Fibrosis is caused by a recessive autosomal mutation in a gene (no idea what the function of this gene was) where patients exhibit a variety of different symptoms (lung infections, poor growth, salty-tasty skin, etc.). In this positional cloning example, the CF gene was cloned with mapping information where a linkage map was built to a known sequence, and chromosome walking and chromosome jumping were also used. Mapping To A Close Known Sequence: -In a diagram of a human chromosome, four markers (M1, M2, M3, and M4) are used in the linkage analysis of a disease phenotype. Each marker provides “linkage coverage” of a portion of the chromosome. This suggests that the gene responsible for the disease lies between those markers. With this information, we could type additional markers that lie between M1 an
More Less

Related notes for Biology 2581B

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.