plan a topics? 1.making a probiotic strain of e.coli that destroys oxalate to help treat kidney...
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Plan ATopics?
1.Making a probiotic strain of E.coli that destroys oxalate to help treat kidney stones in collaboration with Dr. Lucent and Dr. VanWert2.Making plants/algae that bypass Rubisco to fix CO2
3.Making vectors for Teresa Wasiluk’s project4.Making vectors for Dr. Harms5.Cloning & sequencing antisense RNA6.Studying ncRNA7.Revisiting blue-green algae that generate electricity8.Something else?
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Plan AAssignments?
1.identify a gene and design primers2.presentation on new sequencing tech3.designing a protocol to verify your clone4.presentations on gene regulation5.presentation on applying mol bio
Other work1.draft of report on cloning & sequencing2.poster for symposium3.final gene report4.draft of formal report 5.formal report
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Genome Projects
Studying structure & function of genomes
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C-value paradox
Size of genomes varies widely: no correlation with species complexity
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Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”
Step 2 is intermediate: “moderately repetitive”
Step 3 is ”unique"
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Molecular cloning
To identify the types of DNA sequences found within each class they must be cloned
Why?
To obtain enough copies of a specific sequence to work with!
typical genes are 1,000 bp cf haploid human genome is 3,000,000,000 bp
average gene is < 1/1,000,000 of total genome
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Recombinant DNA
Arose from 2 key discoveries in the 1960's
1) Werner Arber: enzymes which cut DNA at specific sites
called "restriction enzymes” because restrict host range for certain bacteriophage
Restriction enzymes create unpaired "sticky ends” which anneal with any complementary sequence
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Recombinant DNA
Arose from 2 key discoveries in the 1960's
1) restriction enzymes
2) Weiss: DNA ligase
-> enzyme which glues
DNA strands together
seals "nicks" in DNA backbone
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Molecular cloning How?1) introduce DNA sequence into a vector• Cut both DNA & vector with restriction enzymes,
anneal & join with DNA ligase• create a recombinant DNA molecule
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Molecular cloning How?1) create recombinant DNA2) transform recombinant molecules into suitable host
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Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules
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Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules
4) Extract DNA
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Vectors
Problem: most DNA will not be propagated in a new host
1) lacks origin of replication that functions in that host
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Vectors
Problem: most DNA will not be propagated in a new host
1) lacks origin of replication that functions in that host
2) lacks reason for host to keep it
DNA is expensive!
synthesis consumes 2 ATP/base
stores one ATP/base
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Vectors
Solution: insert DNA into a vector
General requirements:
1) origin of replication
2) selectable marker
3) cloning site: region
where foreign DNA
can be inserted
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Vectors1) plasmids: circular pieces of”extrachromosomal” DNA propagated inside host•origin of replication•selectable marker (usually a drug resistance gene)Multiple cloning site• Upper limit:
~10,000 b.p. insertsTransform into host
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Vectors
1) Plasmids
2) Viruses
• must have a
dispensable region
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Viral Vectorsfind viruses with a dispensable regionReplace with new DNAPackage recombinant genome into capsidInfect host
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Viral Vectors1) viruses are very good at infecting new hosts
transfect up to 50% of recombinant molecules into host(cf < 0.01% for transformation)
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Viral Vectors1) viruses are very good at infecting new hosts
transfect up to 50% of recombinant molecules into host(cf < 0.01% for transformation)
2) viruses are very good at forcing hosts to replicate themmay not need a selectable marker
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Viral Vectors1) viruses are very good at infecting new hosts
transfect up to 50% of recombinant molecules into host(cf < 0.01% for transformation)
2) viruses are very good at forcing hosts to replicate themmay not need a selectable marker
DisadvantageViruses are much harder to work with than plasmids
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VectorsViruses• Lambda: can dispense with 20 kb needed for lysogeny
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VectorsVirusesReplace "lysogenic genes "with foreign DNA then package in vitro
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VectorsViruses• Lambda: can dispense with 20 kb• M13: makes single-stranded DNA
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VectorsViruses• Lambda: can dispense with 20 kb• M13: makes single-stranded DNA • disarmed retroviruses to transform animals
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VectorsOther viruses• adenoviruses or herpes viruses for gene therapy•Treating patients with engineered viruses that furnish missing genes to specific tissues
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VectorsViruses• Lambda: can dispense with 20 kb• M13: makes single-stranded DNA • disarmed retroviruses to transform animals• adenoviruses or herpes viruses for gene therapy• vaccinia for making vaccines
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Vectors
Artificial chromosomes
Lambda can only carry 20,000 bp
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Vectors
Artificial chromosomes
Lambda can only carry 20,000 bp = 1/150,000 human genome
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Vectors
Artificial chromosomes
Lambda can only carry 20,000 bp = 1/150,000 human genome
need > 750,000 different lambda to clone 95% of entire human genome
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Artificial chromosomes
1) YACs (yeast artificial chromosomes) can carry > 1,000,000 b.p.
• developed for genome projects, but also taught about genome structure
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YACs
• found eukaryotic origins
of replication using
“cloning by complementation”
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YACs• found eukaryotic origins of replication using “cloning by complementation”
randomly add yeast sequences to a selectable marker and transform
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YACs
found eukaryotic origins
of replication using
“cloning by complementation”
randomly add yeast sequences
to a selectable marker and transform
only cells which took up plasmid
containing marker and origin grew
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YACs
found eukaryotic origins
of replication using
“cloning by complementation”
randomly add yeast sequences
to a selectable marker and transform
only cells which took up plasmid containing marker and origin grew call eukaryotic origins ARS = autonomously replicating sequences
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YACs (yeast artificial chromosomes) found yeast centromeres by “complementation cloning ”randomly add yeast sequences to marker & ARS and transformonly cells which took up plasmid containing marker, ARS and centromere grew fast
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YACs (yeast artificial chromosomes) Yeast do not propagatecircles > 100 kBfound yeast telomeres by“complementation cloning ”randomly add yeast sequences to linear DNA with marker, ARS & centromereonly cells which took up linear molecules containing telomere grew
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Artificial chromosomesYACs can carry >1,000,000 b.p.
contain yeast centromeres so that will be transmitted at mitosis contain ARS = origins of replication contain telomeres so that don’t lose ends contain a selectable marker (usually a gene for amino acid or nucleoside biosynthesis)