Chapter 3B

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Chapter 3B

• Recombinant DNA

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Plasmids

• Berg worked with chromosomal DNA
• Cohen worked with small circular pieces of DNA
  known as plasmids
• Found in bacteria
• Extrachromosomal DNA in cytoplasm
• Small range from 1,000 to 4,000 bp in size
• Self-replicating
• Plasmid are used as vectors
• Vectors are pieces of DNA that can accept, carry,
  and replicate other pieces of DNA

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Plasmids

• Some plasmids are represented by 10 to 100 copies
  per host cell high copy number plasmids
  (relaxed plasmids)
• Some maintain 1 to 4 copies per cell low copy
  number plasmids (stringent controlled)
• Naturally occurring plasmids unmodified and
  unengineered plasmids often lack several
  important features that are required for a
  high-quality cloning vector

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Types of Plasmids

• Some plasmids are represented by 10 to 100 copies
  per host cell high copy number plasmids
  (relaxed plasmids)
• Some maintain 1 to 4 copies per cell low copy
  number plasmids (stringent controlled)
• Naturally occurring plasmids unmodified and
  unengineered plasmids often lack several
  important features that are required for a
  high-quality cloning vector

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DNA Ligase

• Cut plasmids can be joined together using DNA
  ligase to create new hybrid (recombinant)
  plasmids
• How does DNA ligase work?
• Catalyses the formation of phosphodiester bonds
  between nucleotides
• Join cohesive ends and blunt ends of DNA
• First plasmid vector was pSC101 contained a gene
  for tetracycline resistance and restriction sites
  for several enzymes

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Cloning vector

• Cohen and Boyer created the first DNA cloning
  vector
• For the insertion and replication of DNA
• Patents received in 1980
• 1974, gene cloning pioneers and cloning critics
  voiced concerns about the safety of genetically
  modified organisms.
• What if it leaves the lab?
• National Institutes of Health formed the
  Recombinant DNA Advisory Committee (RAC) to
  evaluate the risk of recombinant DNA technology.
  Set guidelines in 1976

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Transformation of Bacterial Cells and Antibiotic
Selection

• Cohen made another important contribution to gene
  cloning
• His laboratory demonstrated transformation
• What is transformation?
• A process of inserting foreign DNA into bacteria
• Treated bacterial cells with calcium chloride
• Added plasmid DNA to cells chilled on ice
• Briefly heated cells and DNA mixture
• Plasmid DNA entered bacterial cells
• Replicate with no problem
• Can also use electroporation (brief pulse of high
  voltage electricity to create holes in bacteria

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Transformation and Selection

• Uptake of E.coli of cloned DNA is transformation
• A cell that is capable of taking up DNA is
  competent
• Natural transformations DNA binds to cell wall,
  DNA enters inner compartment that is protected by
  nuclease, linear integrates in host genome,
  circular stay in cytoplasm

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How do you make cells competent?

• Electroporation, needles, and chemicals
• Treat cells to enhance the acquistion of DNA by
  the cell
• Calcium chloride (CaCL2) and heat at 42C
• Adhesion zones or pores in growing cells
• Cold crystallizes the fluid cell membrane,
  stabilizing the charged phosphates. The cations
  form complexes with exposed phosphate groups,
  shielding negative charges- ionic shield so
  plasmid can move through the adhesive zone

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Electroporation

• To insert large circular DNA molecules into host
  cells
• Mix cells and vector together in a chamber and
  apply a pulse of electricity across the chamber.
• Change the electrical properties of the cells and
  the DNA enters
• Insert up to 1 million base pairs (cosmids,
  bacteriophages, etc)
• Used with a wide variety of host cells (fish,
  frogs, etc).

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Screening to find vectors

• How do distinguish nontransformed bacteria and
  bacteria containing the plasmid?
• Use a process called selection
• Selecting for recombinant bacteria while
  preventing the growth of nontransformed bacteria
  and bacteria that contain plasmid without DNA
  insert
• Antibiotic Selection
• Cells are plated with different antibiotics for
  selection

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Blue-white selection procedure

• Plasmid contains AMPr site and Restriction sites
• DNA is cloned into a restriction site in the lacZ
  gene
• LacZ gene encode beta-galactosidase and enzyme
  that degraed lactose into glucose and galactose
• When gene is disrupted, lacZ gene is incapable of
  producing function beta-galactosidase

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• Transformed bacteria are plated on agar
  containing ampicillin
• Nontransformed bacteria can not grow on plates
• Agar also contains chromogenic substrate for
  B-gal called X-gal turns blue when cleaved by
  B-gal
• Nonrecombinant bacteria that contain the plasmid
  but not the insert have a functional B-gal gene-
  Blue
• Recombinant bacteria with insert do not have a
  functional gene- cannot metabolize X-gal - White

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pUC19

• 2686 base pairs, Amp r gene
• Regulatable segment of ?-galactosidase gene (Lac
  Z) of lactose operon of E. Coli.
• Lac I gene that produces a repressor protein that
  regulates the expression of lac Z gene
• Many unique cloning sites multiple cloning
  sites
• Origin of replication

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What makes a Good Vector

• Number of different vectors
• Plasmids are the most popular
• First widely used plasmid DNA vector is pBR322
• Designed to have genes for ampicillin and
  tetracyclin resistance

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Plasmid pBR322

• General purpose plasmid cloning vector
• 4,361 bp
• Two antibiotic resistance genes
• Origin of replication for E. Coli
• High copy number

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Practical Features of DNA Cloning Vectors

• Modern plasmid DNA cloning vectors
• Size small enough to be easily separated from
  chromosomal DNA
• Origin of replication (ori) allows plasmids to
  replicate
• Mulitple cloning site (MCS) restriction sites
• Selectable marker genes ampR or lacZ genes
• RNA polymerase promoter regions important for
  transcription
• DNA sequencing primers for sequencing

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Phage library

• Isolate DNA, cut with RE to generate large pieces
  (partial cut). Use centrifugation to select
  large pieces 20 kb or separate by gel and
  extract.
• Ligate with phage (library), package in E. Coli.
• Phage injects its genetic material into E. Coli
  cell, replicated. Remember phage encodes a
  protein needed to package new viral particles.
• DNA packaged into viral particles and cells lyse
  - E. coli dies-plaque or clear spot is created
  on the surface of the plate (lytic phase)
• Each plaque originates from a single infected
  cell isolate the plaque

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Two important feature of lambda vectors

• Bacteriophage ? vectors-M13 libraries sometimes
  contain large pieces of DNA- can carry up to 25kb
  
• Makes the job of screening more manageable
• Introducing recombinant DNA into E. Coli by phage
  infection is more efficient than transforming E.
  Coli with plasmids

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Southern Blotting

• Used to determine gene copy number
• Developed by Ed Southern in 1975
• Begins with digesting chromosomal DNA into small
  fragments with restriction enzymes
• DNA fragments are separated by agarose gel
  electrophoresis
• Southern blotting is used to visualize specific
  fragments of interest
• Gel is treated with alkaline solution to denature
  DNA
• Fragments transferred to membrane (blotting)

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Setting up the gel sandwich

• Gel is placed under the nylon or nitrocellulose
  membrane, filter paper, paper towels, and weight
  allowed for wicking the salt solution through gel
• DNA is transferred from the gel to the filter by
  capillary action or by current
• Blot is then incubated with nonradioactive or
  radioactive probe.

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Polymerase Chain Reaction

• More rapid approach to identify gene of interest
• Kary Mullis 1980 Nobel Prize in Chemistry for
  his invention.
• Technique for making copies or amplifying a
  specific sequences of DNA in a short period of
  time
• Things you need target DNA to be amplified,
  deoxyribonucleotides, buffer, DNA polymerase, and
  set of primers

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Primers

• Short single-stranded DNA oligonucleotides (20-30
  nucleotides long)
• Complementary to nucleotides flanking opposite
  ends of the target DNA to be amplified.
• Reaction tube place in thermal cycler
• Sophisticated heating block that is capable of
  rapidly changing temperature over very short time
  intervals.
• You go through a series of reactions called the
  PCR cycle

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PCR cycle

• Each cycle consist of three stages
• Denaturation
• Reaction tube is heated to 94-960C causing
  separation of the target DNA into single strands
• Hybridization (Annealing)
• Tube is cooled slightly to 60-650C, which allows
  the primers to hydrogen bond to complementary
  bases at opposite ends of the target sequences
• Extension (Elongation)
• Temperature is raised slightly to 70-75C and
  DNA polymerase copies the target DNA by binding
  to the 3end of each primer.
• At the end of one cycle- target DNA has doubled
  usually run about 30-40 cycles.
• http//www.dnalc.org/ddnalc/resources/pcr.html

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PCR DNA DNA polymerase Primers Denaturation
stage Hybridization/Annealing stage Extension
stage
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Type of DNA polymerase important

• Must use an enzyme that is suitable for the
  various temperature changes.
• Most popular is Taq DNA polymerase. Isolated
  from archaea called Thermus aquaticus, a species
  that is adjusted to hot temperature.
• Named the molecule of the year by the Journal
  Science in 1989.

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Advantage of PCR

• Amplify millions of copies of target DNA from a
  very small amount.
• After 20 cycles approximately 1 million copies
  are produces -2 20
• Real-time PCR used primers with fluorescent dyes
  to quantify amplification reactions.

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Probe

• General Rule sequence of 16 nucleotides or
  longer is likely to be unique in the human
  genome. (exception- repetitive sequence for some
  genes)
• Probe around 100 400 bases is very good
• Complementary base pairing provides the most
  powerful tool for a probe. DNA/RNA and DNA/DNA
• Remember definition of denaturation heat above
  90C and pH above 10.5 (alkaline)
• Renaturation-reduce temperature and pH
• Hybridization annealing

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Properties of probe

• Labeled
• Sequence complementary to gene of interest
• Must be incubated with denatured DNA
• Added in excess to increase the likelihood of the
  gene hybridizing with the probe instead of its
  complementary strand

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Making a probe

• Requires a purified DNA sequence complementary to
  the gene of interest
• In order to identify your target gene, you need
  to already have a piece of that gene or no
  something about the conserved sequence.
• mRNAs within a cell are somewhat unique. mRNA is
  complementary to the DNA sequence of the gene
  from which it was transcribed

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Probe

• Radioactive or nonradioactive probe
• Wash away unbound probe.
• Use photographic film to identify clones with
  gene.
• Autoradiography radioactivity or light
  (chemiluminescence) will expose silver grains on
  the film.
• Compare to original plate to identify clones
• Labeled with a dye

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Genomic Library vs cDNA library

• Goal of Biotechnology is to make protein for
  numerous applications
• Prokaryotes structural gene form a continuous
  coding domain in genomic DNA( rarely have
  introns)
• Eukaryotes structural genes have coding regions
  (exon) separated by noncoding regions (introns)
• Use different strategies to clone these genes
• How would you make a cDNA library

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Prokaryote

• Target gene 0.02 of total chromosomal DNA
• Cut the complete DNA with RE
• Each fragment is inserted into a vector
• Identify specific clone that carries the target
  DNA, isolate, and characterize the gene
• The process of subdividing DNA into clonable
  elements and insertion into host is called
  creating a library (gene bank, clone bank)
• A complete library, by definition, contains all
  the genomic DNA of the source organism.

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Techniques

• Prokarytoes 4 cutter RE 256 bps generated
• Set conditions to give incomplete cuts to
  generate all the possible fragment sizes
• Low concentration of RE
• Shorten incubation time
• Sum of clone 3X more than the amount of genome
• 4 x 10e6 bp size for prokaryotic genome.
  Approximate size per piece 1000 by
• 3 ( 4 x 10e6 bp)/ 1000 12,000 clones

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Eukaryotic genome

• 3.3 x 10 9 bp
• Large pieces bacterial artificial chromosome
  clones
• 80,000 clones each with 150,000 bp insert

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Vectors for Cloning Large Pieces of DNA

• Bacteriophage ? vectors libraries sometimes
  contain large pieces of DNA
• E. Coli virus engineered to be a vector for
  inserts in the range of 15 to 20 kb. Replacing
  20 kb of cloned DNA with 20 kb of lambda DNA

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Cosmids, YACS, and BACS

• Cosmids
• Carry about 45kb of cloned DNA and maintained as
  plasmids in E. Coli.
• Combine the properties of plasmids and
  bacteriophage lambda vectors
• pLFR-5 has two cos sites, RE site, MC site, etc
• YACS-yeast artificial chromosome, accepts 1000kb
  inserts
• BACS-bacterial artificial chromosomes, accepts
  1000kb inserts

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Southern Blotting and PCR

• http//highered.mcgraw-hill.com/olcweb/cgi/pluginp
  op.cgi?itswf535535/sites/dl/free/0072437316
  /120078/bio_g.swfSouthernBlot
• http//www.sinauer.com/cooper/4e/animations0411.ht
  ml
• http//www.maxanim.com/genetics/PCR/PCR.htm