Recombinant DNA Technology

1
Recombinant DNA Technology

• Chapter 19

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Recombinant DNA Technology

• 1971 paper by Kathleen Dana and Daniel Nathans
  described isolation of enzyme that cleaved DNA at
  specific sequences
• 1978 Nobel Prize to Nathans, Smith and Arber for
  restriction endonuclease discovery

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Cloning DNA Molecules

• Purify DNA from desired source
• Cut DNA with restriction endonuclease
• Join fragments to cloning vectors cleaved with
  compatible restriction endonuclease to create
  recombinant DNA molecules
• Transfer recombinant molecules to host cells
• Isolate DNA from individual clones of transformed
  host cells
• Do as you please with the isolated DNA segments

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Restriction Endonucleases

• Hundreds now identified
• Host cell defense against invading DNAs
• Cleave DNA at or near a specific recognition
  sequence
• Creates restriction fragments
• Recognition sites can be from 4 to 8 base pairs
  and are commonly palindromes
• First one isolated was EcoRI from E. coli
• Often produce sticky ends

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Restriction Endonucleases
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DNA Cleavage By EcoRI
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Recombinant DNA Molecules
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Cloning Vectors

• Plasmids
• Phage
• Cosmids
• BACs
• YACs

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Plasmids

• Circular independent replicons, origin of
  replication (ori)
• Generally encode useful but not essential genes
• E.g. antibiotic resistance or catabolic pathways
• Allow cloning fragments up to about 10 kbp
• Selectable markers
• Multiple cloning sites

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Plasmid Cloning Vectors

• As small as possible with minimum restriction
  endonuclease cutting sites in genes
• ori
• Selectable marker(s)
• Multiple cloning site (MCS)
• Reporter function useful

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lacZ Complementation

• Plasmid has portion of lacZ gene flanking MCS
• Host strain has lacZ gene missing this seqeunce
• Expression of two components still yields a
  functional LacZ
• Like it has 2 subunits
• Cloning into MCS eliminates complementation
• X-gal in media allows for blue/white selection

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Bacteriophage Vectors

• Commonly based upon l phage
• Most internal genes deleted
• Insert DNA into middle region (up to10-15 kb)
• Package
• Infect host cells with integrated helper phage to
  provide missing protein-encoding genes

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Cosmids

• Plasmid with l phage packaging sequence (cos)
• Can clone up to 50 kb
• Packaged into l particles and injected into host
  cells
• Circularizes in cell and continues as a large
  plasmid

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BACs

• Bacterial artificial chromosomes
• Can clone up to 200 kb DNA fragments
• Based upon F plasmid
• Origin, selectable marker, promoters to expressed
  cloned genes

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YACs

• Yeast artificial chromosomes
• Have centromere, telomeres and an origin of
  replication, plus selectable markers
• Cloned segments of 250 kb

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Expression Vectors

• Also include regulatable high level expression
  promoter
• T7 phage promoter
• lac operator
• lac repressor gene

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First Prokaryotic Host Cells

• First clones introduced into strains of E. coli
  K12
• Protocol
• Isolate target DNA
• Cut with RE
• Ligate to vector
• Transform host cells
• Plate on antibiotic-containing medium
• Identify recombinant plasmids
• Identify/characterize specific clones

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Cloning into Plasmids
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Expression of Recombinant Genes in Eukaryotes

• Expression is sometimes desirable in eukaryotic
  cells
• Especially if post-translational modifications
  are important or study simply requires it

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Eukaryotic Expression Vectors

• Generally similar to prokaryotic ones
• Commonly start with prokaryotic vector
• Construction done in E. coli
• Shuttle vector
• Origin
• MCS
• Selectable marker
• High expression regulatable promoter
• But also generally an intron included
• Especially for cDNA clones

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Cloning into Plant Cells

• Vectors based upon Ti plasmid
• Derived from Agrobacterium tumifaciens plasmid

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Cloning into Mammalian Hosts

• Especially for model systems
• DNA integrates into a host cell chromosome
• Random vs. site-directed
• Transgenics
• Models for diseases
• Improved individuals???

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Transgenic Mammals
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Polymerase Chain Reaction

• PCR
• 1993Nobel Prize for Kary Mullis
• Proposed in 1986
• Can provide millions of fold amplification of a
  DNA fragment or sequence
• Needle in haystack
• Revolutionized molecular biology/genetics/forensic
  s/and everything
• Day earth changed

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PCR Procedure

• Denature DNA
• Add primers, thermostabile DNAP, dNTPs
• Extension
• Denaturation
• Bind primers
• Extension
• Repeat last 3 steps thirty times

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Chromosome Sorting

• Important for early portion of human genome
  project
• Simplified sequencing effort
• Fluorescent tags on specific chromosomes

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Pulse Field Gel Electrophoresis

• For very large DNA molecules
• To left are intact yeast chromosomes
• Electric field is pulsed/changed after very short
  intervals

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cDNAs

• Copies of mRNAs in DNA
• By reverse transcriptase
• Needed to analyze genes and also to express
  eukaryotic genes in prokaryotic systems
• Introns
• cDNA libraries

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cDNA Synthesis

• Oligo T primer
• Reverse transcriptase and deoxyribonucleotides
• RNaseH
• DNAP I makes second strand using the hairpin
  created by reverse transcriptase as a primer
• Hairpin cleave by S1 nuclease

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Library Screening

• General approach used for both genomic and cDNA
  libraries
• DNA from colonies/plaques
• transferred to membrane
• Denature
• Hybridize with probe
• Detect probe binding
• Method depends upon labeling of probe
• Isolate specific probe-binding clones and culture

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Restriction Maps

• Useful but now most commonly generated by
  computer from actual DNA sequence

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Southern Transfer

• Developed by E. M. Southern
• Method for transferring DNA from a gel to a
  membrane
• Described to right
• Also Western and Northern blots
• Proteins and RNA

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Hybridization

• Binding of a probe (generally RNA or DNA) to a
  nucleic acid in a gel or more commonly bound to a
  membrane

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DNA Sequencing

• Sanger
• Method described to right

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Chain Termination DNA Sequencing

• Autoradiograph of results from Sanger
  dideoxyribonucleotide chain termination method
• Sequence is read from bottom to top

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Fluorescent Dyes

• DNA sequencing with fluorescent dyes attached to
  chain terminator dideoxyribonucleotides
• Allows for automated DNA sequencing

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Electropherogram