Applications or Recombinant DNA Technology

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Chapter 13

• Applications or Recombinant DNA Technology

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Rec. DNA techniques very powerfulwhat can we
do?? We will discuss

• Mutagenesis in vitro
• RFLP Mapping
• Reverse Genetics
• Expressing eukaryotic DNA in bacteria
• Rec. DNA in eukarya
• Transgenic eukaryotes
• Yeast
• Plants
• Animals
• Gene Therapy
• Using Rec. DNA to detect disease alleles directly

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• In vitro mutagenesis designer genes.
• Create any mutation you wish in a cloned DNA
  target
• Uses oligonucleotides (oligos) with desired
  mutation to hybridize to a given target
• Oligo primes DNA pol synthesis to duplex form and
  transform into bacterial cell
• Grow up and sequence clones to find your
  mutation.
• Possible to make point mutant, deletion,
  insertion.

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Targeted mutagenesis
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Restriction fragment length polymorphisms
Some individual variation of genomic sequence in
population easy to see with RE on Southern
blots of genomic DNA.
E
E
E

• NOTE Treat RFLPS like alleles
• -Presence of a RE site is a plus
  ()-Absence of a RE site is a minus (-)
• RFLPS are pretty common!
• How to find? Hit or miss process of hyb. Random
  clones to RE digested DNA from individuals at
  random or in a single family. WHY USE RFLPS?
• RFLPS can define a locus that is heterozygous
  morph can be used as a neutral marker in chrom.
  Mapping.
• RFLPs are useful diagnostic tools with genetic
  diseases if you can find a RFLP that always seems
  to associate with the disease allele, the RFLP
  can be diagnostic marker for that particular
  genetic disease.
• Comparing RFLPs measures genetic
  diversification, hence evolution

2KB
Person A
Probe
Silent change (point mutant) at this site Site
lost
E
E
2.3 KB
Person B
Probe
Genome B
Genome A
MW markers
This defines two morphs, one is 2 kb other is
2.3 kb
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• Good example of inheritance of a RFLP showing
  linkage to dominant disease allele D
• Note RFLP maps based on recombination analysis
  of mating.
• RE maps based on physical analysis of DNA

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Reverse Genetics vs. Genetics
Genetics mutant phenotypemutant alleleDNA
seq.ptn seq. Reverse ptn seq. DNA
seq.mutate allele evaluate mutant
phenotype Ptn in search of a functionGene in
search of a phenotype
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• Example of Reverse Genetics at work
• ORF (from computer) In vitro mutagenesis back
  in organism Results??
• Amino acid sequence back translate ORF to DNA
  make probe to find gene clone it, mutate put
  back to see results
• Important tools in the process Gene disruption
  or gene knock outs.

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How to get a vector inside a cells to make a
transgenic organism?
BANG!
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Tools to allow expression of eukaryotic genes in
bacteria. Expression Vectors factories for ptn
production in a robust system (like E.
coli) (Defines the term biotechnology which is
commercialization of recomb. DNA) -Patented life
forms now possible. -Good example T7 RNA pol.
2 step overexpression system for Factor VIII.
2 plasmids Induce Rpol with IPTG inactivates
the lac repressor
Very high copy plasmid
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Many other examples of overexpression of useful
gene products in bacterial systems
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Another variation This represents a great way
to get the ptn OUT of the bacterium
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Recombinant DNA in Eukaryotes
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Basic Cloning established in bacterial systems.
Now applicable to eukaryotes. Eukaryotic
expression systems high value since E. coli is
NOT ideal for higher organism expression
since -Post Tln modification not possible -Ptn
degradation -Problems with extraction/yields and
purification -bacteria may be a hostile
environment An excellent system Baculovirus.
A DNA insect virus
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Very large genome of 50 kb
Coat gene promoter very robust, expressed LATE
in infection (therefore not essential for
productive infection)
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Transgenic Eukaryotes

• DNA introduced in Euk. Cells in many ways (13-10)
• Integration typically (a few ARS exist)
• Good for promoter analyses
• Plants, animals, fungi large commercial value
  with all these species.
• Great increases in scope of field

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Saccharomyces cerevisiae (aka bakers yeast)

• The most exciting and sophisticated euk. Model
  system
• Fully sequenced genome
• Good understanding of transmission genetics and
  life style
• Many Many strains, plasmids, mutants, cool stuff,
  etc.
• Specific plasmid 2 micron plasmid (6.3 kb
  natural plasmid) forms basis of most vectors in
  yeast
• Plasmid can be an ARS or integrative (targeted)
• Targeted integration BIG value added in yeast
• NEXT details of 2 u plasmids

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Yeast 2 uM Plasmids
Simplest integrates into genome, derived from
bacterial plasmids (single or dbl crossover).
Bacterial vector based, so no replication
possible forces integration (selection with a
marker, like ura

• Shuttle vector pass through yeast AND E. coli
  and have high utility in routine cloning
• ARS do not ALWAYS segregate to daughters at M
  thus, add a yeast centromere as shown in c
  (attachment site for nuclear spindle ensures
  proper partitioning and behaves like a
  chromosome).
• To make a real chromosome should linearize DNA
  and add telomeres at ends to give a YAC (d).
• YACs behave like minichrom. In yeast and show a
  22 segregation from diploids.

The basic 2- uM vector basis for shuttle in a.