1. Material for Quiz 5 from Chapter 8

2. Will not cover ID of clones in expression libraries
Screening for Specific Genes in a Collection of Recombinant Clones
Collection = Library = all possible DNA sequences present in at least one clone in the collection = “complete” library
Genomic library
cDNA library
Vector depends on size of insert needed and size of library needed
Method used to screen (search) for desired clone depends on information available
Steps in the screen:
Plate bacteria/virus on petri plates (colonies/plaques)
Make replica of plates on filters: DNA from clones exposed and denatured on the filters
“Probe” filter to identify specific clone
Pick colony/plaque identified by probe, amplify in bacteria, analyze use as desired…
Probes for specific genes:
32-P labeled DNA if DNA sequence of gene or amino acid sequence of protein is known
oligonucleotides synthesized in vitro (commercially available
homologous gene
PCR product
Antibody to protein product
Vector must be an “expression vector” where the cloned gene is expressed in E. coli

3. Positional Cloning and Chromosome Walking
Positional Cloning of genes when all you know is the identity of genes nearby (linked)
Disease gene is linked to A
Identify clones from library that contain A
“Walk from A to D using the clones identified in the screen for A…

4. Cloning by “Gene Tagging”
Create mutant for gene of interest in an experimental organism by insertional inactivation
Example method: transposon tagging
Activate a transposon in an organism
Screen for mutants that show the phenotype predicted
Make genomic DNA library using DNA from the mutant
Screen for clones containing the transposon

5. Applications of Recombinant DNA: Prenatal Screening
Applications of Recombinant DNA Technology
Early detection of disease associated alleles
Amniocentesis
Chorionic villus sampling
PCR amplify region of interest and analyses (mutant or wild type)

6. Southern Blot Analysis
Restriction Mapping of Genomic DNA by Southern Blot analysis
What is the map of the chromosome near gene A?
Cut total genomic DNA with Enzymes (E = EcoRI, B=BamHI, H = HindIII)
Electrophorese DNA through gel along with molecular weight markers
Transfer DNA from gel to a filter
Filter is a replica of pattern of DNA in the gel
Probe with 32P-labelled DNA probe (gene A)
Autoradiography – only DNA bands that hybridize to probe will be detected
Gene A is on a 4 kb EcoRI frgment, a 3 kb BamHI fragment, and a 7 kb HindIII fragment
What if double digests were performed?
EcoRI plus BamHI = same pattern as BamHI,
EcoRI plus HindIII = same pattern as EcoRI
HindIII plus BamHI = same pattern as BamHI
Where are the EcoRI, BamHI, and HindIII sites relative to gene A?
What if you were screening for a deletion of gene A as part of early detection of disease (aminocentesis or chorinic villus sampling)?
E B H MW
markers

7. Applications of Recombinant DNA: Genetic Engineering
Create genetically engineered organisms to study gene function
Transgenes and transgenic organisms
Ectopic insertion of transgene (extra copy of gene; used to determine whether the gene you cloned can complement (fix) a mutation
Did you really clone the gene you were looking for?
Targeted Gene insertion
Replacement of endogenous gene with genetically engineered gene
Example:Gene Knock out in Mice (to determine the function of a gene)
Gene Knockout in Mice
Knockout Construct:
Flanking DNA to direct homologous recombination
Gene of interest mutated in some way (usually by insertion into or replacement of an exon with a selectable marker = neomycin resistance – SELECT FOR neoR)
Second marker genes that you can SELECT AGAISNT (ganciclovir sensitivity)
Method:
Transform ES cells of AA mouse – select neoR and Ganciclovir resistant = A/A; M/m cells
Inject A/A M/m cells into embryo = a/a M/M
Transplant embryo into a/a M/M mother
Look for a/a M/M + A/A M/m chimeras (by agouti phenotype)
Cross chimeras to a/a M/M
Identify progeny that are M/m by DNA analysis
Cross two M/m mice to get m/m mice and determine the phenotype

8. Applications of Recombinant DNA: Genetic Engineering
Create genetically engineered organisms to study gene function
Transgenes and transgenic organisms
Ectopic insertion of transgene (extra copy of gene; used to determine whether the gene you cloned can complement (fix) a mutation
Did you really clone the gene you were looking for?
Targeted Gene insertion
Replacement of endogenous gene with genetically engineered gene
Example:Gene Knock out in Mice (to determine the function of a gene)
Gene Knockout in Mice
Knockout Construct:
Flanking DNA to direct homologous recombination
Gene of interest mutated in some way (usually by insertion into or replacement of an exon with a selectable marker = neomycin resistance – SELECT FOR neoR)
Second marker genes that you can SELECT AGAISNT (ganciclovir sensitivity)
Method:
Transform ES cells of AA mouse – select neoR and Ganciclovir resistant = A/A; M/m cells
Inject A/A M/m cells into embryo = a/a M/M
Transplant embryo into a/a M/M mother
Look for a/a M/M + A/A M/m chimeras (by agouti phenotype)
Cross chimeras to a/a M/M
Identify progeny that are M/m by DNA analysis
Cross two M/m mice to get m/m mice and determine the phenotype