Genetics Notes week 10
Genetics Notes week 10 Bios 206
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This 3 page Class Notes was uploaded by Becca Sehnert on Friday March 18, 2016. The Class Notes belongs to Bios 206 at University of Nebraska Lincoln taught by Dr. Christensen in Fall 2016. Since its upload, it has received 13 views. For similar materials see Genetics in Biological Sciences at University of Nebraska Lincoln.
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Date Created: 03/18/16
WEEK 10 MONDAY Ch 20, Recombinant DNA • Recombinant DNA refers to joining DNA molecules, usually from different sources, that are not found in nature o Cut DNA with restriction enzymes –from bacteria o Ligate to a vector –ligase o Transfer to a host cell o Let host replicate DNA o Recover DNA from host cell • Restriction enzymes cut DNA as specific sequences o Restriction enzyme binds to DNA at specific sequence and breaks phosphodiester bonds o Most recognition sequences are palindromic –symmetrical, 5’ to 3’ o Restriction enzymes cleave sequences at staggered positions in recognition sequence Which of these DNA sequences is palindromic when double stranded a. it was the C answer • Ligation o DNA ligase joins reseriction fragments covalently to produce intact DNA molecules o Blunt ends can be ligated, but sticky ends work better • Vectors o DNA molecules that can replicated cloned DNA fragments in a host cell o Vestors must be able to replicate independently (origin of replication) o Vectors should have several restriction enzyme sites to allow insertion of DNA o Vectors must have a selectable markers such as antibiotic resistance § Vampires at a night game • Hold up a mirror –screen • Don’t invite them in –counter selection • Kill everyone with a lead bullet –selection , vamps don’t die § Natural selection works the same way • Plasmids o Extrachromosomal double-stranded DNA molecule that replicates autonomously in bacterial cells o Plasmids used for DNA cloning usually have been engineered to contain: § # of convenient restriction sites § Marker gene to select for its presence in host cell § Replicating continuously, amplified it o Selection for cells that have it, and not for cells who don’t o Screen and selection all in one plate (color dying the DNA molecules) • Bacteriophage vectors o Large chunk of DNA o Used to be very important o Central third of lambda phase vectors can be replaced with foreign DNA • Cosmids o Hybrid of lambda phage and parts of plamids o Have advantage of being able to be packaged into phage for efficient delivery to cells Why is ampoicillin resitance gene present in many vectors? a. So cells without plasmid will die b. To detect which plasmids have insert of foreign DNA c. To make colonies turn blue d. So plasmid will replicate to high copy number • BAC’s o Bacterial artificial chromosomes (BAC) based on F factor and can carry up to 300 kb of inserted DNA o Low copy number • Expression vectors o Drive expression to make lots of encoded protein o Available for bacterial and eukaryotic hosts o Lots of reasons to make lots of one protein • Other eukaryotic hosts o In addition to bacteria and yeast, plant and animal cells can serve as hosts for recombinant DNA • Transforming plant cells o Agrobacterium tumefaciens used to transform plant cells with T-RNA containing foreign DNA o T-DNA and insert integrate into plant genome o Plant cells can be grown in tissue cultural FRIDAY The goose alchemase gene is under study. In Canadian geese, the mutation inactivates the gene is an 800 base pair transposon inserted in location shown in diagram below. Sites for restriction enzyme EcoRI and Sall are shown in the map, along with distances between adjacent sites in kilobases. Two different probes are available. EcoRI and Sall cut at different sites You want a probe that will tell you something useful You got a bunch of fragments *Wild dominant Which probe and restriction enzyme would you use in a Southern blotting experiment to see whether the mutation is present or not? a. Probe 1 and EcoRI b. Probe 1 and Sall –don’t pick this one c. Probe 1and EcoRI d. Probe 2and Sall e. Probe 2and both EcoRI and Sall –this is wrong Remember you want a goose with an active alchemase gene. Using Probe one and EcoRI on a southern blot, what fragment on blot would tell you to buy the goose? a. 5.8 kb b. 6.6 kb –you don’t want the transposon in there c. 3.0 kb d. 2.0 kb e. 2.8 kb Northern lots are used to determine whether gene is actively being expressed in given cell or tissue. • Similar to Southern blotting, but northerns use RNA on gel, separated by size DNA sequencing is ultimate way to characterize a clone • Dideoxy chain termination sequencing, developed by Sanger o All end in A • Large scale genome sequencing is automated band uses fluorescent dye- labeled dideoxy-nucleotides § Everytime polymerase adds an enxyme • Third Generation Sequencing –figure 20-17 •