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molecular cloning
isolating a specific gene from the genome and making many copies, usually by propagating it in a plasmid
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why do molecular cloning
- used to understand the function of the gene:
- move gene into a different organism, different mutant background
- change its regulation
- specifically mutate that gene
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basic steps in molecular cloning
- cut foreign DNA (can be chromosome if target gene is unknown or PCR product if target gene is known) and vector with same restriction enzyme to create complimentary sticky ends
- add DNA ligase to form recombinant molecules
- introduce recombinant vector into a host using transformation or conjugation
- select transformants that are resistant to vector antibiotic marker
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what are restriction enzymes and why do bacteria have them?
- protect from foreign DNA (often phage genomes)
- restriction endonuclease cuts specific, palindromic DNA sequence, they are dimers with two active sites
- modification systems - methylase protects host DNA by methylating a base within the same sequence, blocking the restriction enzyme
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vector
- plasmid used to carry your gene of interest
- have multiple cloning sites or polylinkers containing several unique restriction enzyme sites for insertion of a DNA fragment
- designed to help you to help you distinguish empty vectors from those containing inserted DNA
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alpha-complementation (blue/white screening)
allows selection of transformants containing a vector and screening for vectors that contain an insert
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background for understanding alpha-complementation
- host cells makes nonfunctional B-gal (white) empty vector - encodes missing a-peptide that restores B-gal activity (blue)
- inserting a fragment into the multiple cloning site of a vector interrupts the coding sequence of the alpha-peptide
- transformants with vectors containing foreign, inserted DNA will remain B-gal-, while those contain empty vectors will become B-gal+
- detect beta-galactosidase activity using an indicator called X-gal, which creates a blue color when it is cleaved by B-gal
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requirements for plasmids - they are designed to work with specific strains
- host DNA replication machinery must recognize plasmid origin of replication
- host strain must be sensitive to antibiotic you are using for selection
- for blue/white screening, host strain must synthesize only the truncated form of B-gal
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How do you identify the affected gene in a mutant?*
complementation of mutant phenotype using genes carried on plasmids - used for identification in random mutagenesis
- If recessive mutation:
- Make genomic DNA library from wild-type parent
Transform mutant with the library
select for presence of plasmid (antibiotic marker) and growth at non-permissive restriction (ex high temperature)
colonies that grow should contain a vector holding the wild-type gene
re-isolate plasmid and sequence the DNA
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How do you identify the affected in a mutant if the mutation is a dominant mutation?
- transform with genomic library and select for transformants on antibiotic medium
- replica plate onto identical plates and grow one at permissive temperature and other at non-permissive temperature
- screen for transformants that grow at permissive temperature sequence insert that was cloned into vector from each transformant
- compare the vector sequence obtained with the sequence from the wild-type chromosome to identify the mutant
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When do you use random mutangenesis and what can you use?
- when i don't know at all what genes are involved in my process of interest
- have no sequence information about my organism
tools - chemical mutagens, UV radiation, transposons
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When do you use targeted mutagenesis and what can you use?
- when I can predict what genes will be involved in my process of interest
- you must have sequence information about the genes you are targeting or a whole genome sequence
tools - targeted gene disruption to create knockout mutations
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How do you create knockout mutations by targeted gene disruption?
- clone gene into a plasmid
- cut gene with restriction enzymes
- replace internal region with antibiotic resistance gene
- leave 500-1000 bp at each end so homologous recominbation can occur between sequences and identical ones at chromosomal locus
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What can you do to ensure recombination with vector in targeted gene disruption?
- linearize plasmid before transformation
- used a vector that cannot replicate in the host
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forward genetics vs. reverse genetics
- forward:
- make random mutations (easier)
- select or screen for mutants with desired phenotype
- reverse:
- make directed mutations in genes of choice (harder)
- test each mutant in collection for desired phenotype
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transposon mutagenesis
- have to mutagenize at random
- select mutants that received a transposon hop
- select or screen for mutants with your desired phenotype
- does not gurantee a mutation in a specific gene
- can insert into any DNA sequence
- do not use homologous recombination
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