Ch 1. My Q's Flashcards
Largest stretch that can be sequence?
600-800bp
How did they increase landmark density in maps?
development of other variable molecular markers (not just RFLPs)
Solutions to the human genome project
ave milestones (genetic map, physical map), sequence model organisms for comparison, develop strategies (automated, computer databases)
Why isolate single genes?
Learn from sequence (is it normal?), Modify genes to study function (knockouts), Move genes between organisms (transgenics)
What’s cDNA
complementary DNA synthesized from single stand RNA (reverse transcription
How can we isolate a gene from within DNA
Extract total DNA (or cDNA) from organism under study —> Cut into pieces (restriction enzymes -RE) –> Isolate a vector and open it up using RE –> Insert DNA pieces into the vector (Recombinant DNA). –> Insert in a cell system (e.g. bacteria) that produces a colony of genetically identical progeny (clones). –> Identify clone carrying the DNA piece of interest
What are Restriction enzymes?
Enzymes that recognize and cut specific 4 to 6 palindrome sequences of DNA. Fragments with sticky ends can be ligated to vectors.
3 things to make a cloning vector
origin of replication (to amplify donor DNA), selectable marker, At least one unique restriction enzyme cleavage site
Plasmid
small circle double stand (dsDNA), ORI, some antibiotic resistance maker, restriction sites
How is vendor and donor DNA sealed?
DNA ligase Phosphodiester bonds at junctions It can also be used on blunt ends
Selection of recombinant clones
Use RE to cut the plasmid and the DNA, (plasmid at TetR) —>ligate the two together—> grow on ampicillin which both have resistance too—> make a stamp of each plate and regrow, one on amp and one on tet. Only non-recmombinate will grow on txt because no foreign DNA.
3 other vectors
Viruses (infect cells with high efficiency. Can take fragments longer than 10Kb. Remember that some eukaryotic genes can be quite long. ) Cosmids (can carry up to 45Kb long donor DNA. They can replicate as plasmids and can be packaged into phage heads. ) Artificial chromosomes ((BACs, YACs) can carry longer fragments of DNA (cloning full genomes).)
What’s a DNA library
A collection of clones, each containing a different sample of the donor DNA of interest.
How to use probe
Prove is a piece of DNA that searches for a sequence within the clone. Plasmid vector—>Trasform Ecoli cells—>velvetten stamp—>Filter removed. Bacteria lysed. DNA denatured and bound to filter—>labeled pride solution added—>Detect hybridization by autoradiography or radiolabeled prove and chemiluminescent election for non radiolabled probe. Dark spot=clones detected by probe
Using cloning to isolate disease-alleles
The disease is inherited together with an allele of a random molecular marker very often if RMM is close by. Ex: Cistic Fibrosis is found to intagrate with 2 MM (MET - D7S8) 98% of the time therefore it’s within he 2.5Mb distance between said markers.
