The second midterm exam to be held on Tuesday, April 1 in class. It covers from the February 13 through the March 25 lecture. This corresponds to pages 174 to 334 (end of chapter 9) in the text. There will be a question and answer session from 4 to 6 PM on Friday, March 28 in Heald Auditorum.
The aspects of genetic engineering to be discussed are cloning of DNA, production of useful substances by genetic engineering, genetic manipulation of plants and animals and genome analysis.
The basic process of cloning DNA involves: (1) identifying a target DNA sequence to be cloned, (2) using a vector to amplify the target DNA, (3) joining the vector and target DNA, usually after both are treated with a restriction enzyme (4) introducing the clone into a host cell for amplification, (5) growing multiple copies of the clone, and (6) identifying clones of interest from a "library".
The principal types of target DNA are genomic DNA and cDNA. CDNA is complementary DNA, prepared using reverse transcriptase enzyme from messenger RNA.
There are several types of vectors. The principal types are plasmids (for inserts up to 10 kb), phage vectors (for inserts 12-20 kb) and cosmids (for inserts 40-45 kb). Artificial chromosomes such as PACs, BACs and YACs can be used for inserts over 100kb in size.
Vectors are often engineered to facilitate the cloning process. They often have "multiple cloning sites", which allow different restriction enzymes to cleave a specific area on the vector. Vectors often have antibiotic resistance, which facilitates selecting for cells carrying them during the cloning process.
Selection of clones carrying inserts may be based on size constraints or on expression. An example of selection based on size constraints is that lambda vectors require an insert in order to package properly. An example of selection based on expression is that many plasmids express the lac Z gene if the cloning site does not carry an insert but do not if they have an insert. A staining reaction results in blue colonies if lacZ is active and white if it is inactive (plasmid carries an insert).
A "library" contains a diversity of clones from the target source. This may consist of bacterial colonies arrayed on a plate, or phage plaques on a lawn of bacteria. Specific sequences may be selected based on colony or plaque lifts followed by hybridization of a probe to the membrane (similar to Southern blot technique).