Exploring Genes and Genomes

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Chapter 5

• Exploring Genes and Genomes

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Basic Tools of Gene Exploration

• Restriction Endonucleases
• DNA manipulation
• Blotting Techniques
• Separate and characterize DNA
• DNA Sequencing
• Gene architecture gene expression, protein
  structure
• Solid-Phase Synthesis of Nucleic Acids
• Identification and amplification of other nucleic
  acids
• Polymerase Chain Reaction
• Amplification of nucleic acid

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Restriction Endonucleases

• Recognize specific base sequences in duplex DNA
• Palindromic inverted repeats
• Site-specific cleavage of duplex DNA
• Cleavage sites symmetrical
• Staggered or even cuts
• Prokaryotes
• 3-letter abbreviation of host, strain number if
  needed, and roman numeral
• Prokaryotes self DNA is not cleaved
• Recognition site methylated in self DNA

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Restriction Digest Mapping

• Separate restriction fragments by gel
  electrophoresis
• Resolving power 1 bases
• Polyacrylamide fragments less than 1000 bp
• Agarose fragments between 1 kb 20 kb
• PFGE agarose pulse electric field - mb
• DNA bands visualized in gel by autoradiography or
  staining
• Radiolabel
• Fluorescent dye ethidium bromide, picrogreen

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Blotting Techniques

• Southern Blotting (DNA Blots)
• Transfer of DNA to a solid support for
  identification and characterization
• Northern Blotting (RNA Blots)
• Transfer of RNA to a solid support for
  identification and characterization
• Western Blotting (Protein Blots)
• Transfer of protein to a solid support for
  identification and characterization
• Probe with labeled antibody

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Southern Blotting Technique

• Restriction digest of duplex DNA
• Electrophoresis to separate fragments
• Denature duplex DNA to single stranded DNA
• Transfer ssDNA to nitrocellulose membrane
• Hybridize with labeled probe containing a
  complementary sequence
• Detection of hybridized probe

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DNA Sequencing

• De novo synthesis of DNA
• Sanger Dideoxy Method
• DNA polymerase for synthesis needs primer
• Controlled termination of replication
• 2,3-dideoxy analog of one of the nucleotides,
  different for each reaction mixture
• Four reaction mixtures containing labeled dNTPs
• Separation of four reactions using
  electrophoresis
• Base sequence read from autoradiogram
  (fluorescence scan) base read is complementary
  to 2,3-dideoxy analog

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Synthesis of DNA

• Automated Solid-Phase Methods
• Growing chain linked to an insoluble support
• Activated monomers sequentially added to growing
  chain
• The desired product remains on the solid support
  until the final release step
• Reactions take place in single vessel and excess
  reagents can be added to drive the reaction to
  completion
• After each step soluble reagents and by-products
  are easily removed from the immobilized growing
  chain

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Automated Solid-Phase Synthesis of DNA

• The 3-OH of a dNMP is immobilized on a glass
  bead. The amino groups on the bases are blocked
• An incoming activated monomer is joined to the
  5-oxygen atom of the immobilized dNMP via the
  3-OH

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Automated Solid-Phase Synthesis of DNA

• The 5-OH of the activated monomer is blocked
  with DMT (dimethoxytrityl)
• The 3-P of the activated monomer is blocked with
  ?CE (?-cyanoethyl group)
• Amino groups on the bases also blocked

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Automated Solid-Phase Synthesis of DNA

• Coupling of activated monomer to growing showing
  performed under anhydrous conditions to prevent
  the reaction of water with the phosphoramidites

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Automated Solid-Phase Synthesis of DNA

• Phosphite triester is oxidized by iodine
• The DMT protecting group on the 5-OH of the
  growing chain is removed by dichloroacetic acid
• Dichloroacetic acid leaves the other protecting
  groups intact

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Automated Solid-Phase Synthesis of DNA

• Each cycle takes about 10 min and elongates more
  than 98 of the chains
• At the end of synthesis, ammonia is used to
  remove all protecting groups and to release the
  oligonucleotide from the solid support
• The strands are of diverse length and can be
  purified using polyacrylamide gel electrophoresis
  or HPLC

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Applications

• Primers
• Label
• PCR
• Protein Engineering
• Tailor made genes
• Produce new proteins with novel properties in
  abundance

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Polymerase Chain Reaction

• Amplify specific DNA sequences
• Need to know flanking sequence of target
• Reaction
• Pair of primers that hybridize to the flanking
  sequences of the target
• dNTPs
• DNA polymerase heat stable

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Polymerase Chain Reactions

• Strand Separation
• 95oC for 15 s
• Hybridization of Primers
• 54oC
• Primer in excess
• Parent duplexes do not form
• DNA Synthesis
• 72oC
• Both strands 5 to 3 direction

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Polymerase Chain Reactions

• Target Sequences
• 3-TAGCGTACG-5
• 5-ATCGCATCG-3
• Primer Sequences
• P1 5-ATC
• P2 5-CGA

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATC
• AGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATC?
• ?AGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCG
• TAGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCGC
• GTAGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCGCA
• CGTAGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCGCAT
• GCGTAGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCGCATGC
• TAGCGTAGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• 3-TAGCGTACG-5
• 5-ATCGCATGCXXXXXXXXXXXXXXX
• XXXXXXTAGCGTAGC-5
• 5-ATCGCATCG-3

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Polymerase Chain Reactions

• Target sequence can be unknown as long as
  flanking sequence information is known
• Target sequence can be larger than primer
• 10 kb
• Primer sequence doesnt have to match exactly the
  flanking sequence
• PCR is highly specific
• Stringent conditions
• PCR is highly sensitive
• Amplify a single molecule

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PCR Application

• Diagnostic in medicine
• Detection of bacteria and viruses
• Forensic
• DNA profile
• Highly variable loci within a population
• Analysis of blood stains and semen samples
• Ancient DNA

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Recombinant DNA Technology

• New combinations of unrelated genes constructed
  in the lab
• Cloned
• Amplified
• Transcribed
• Translated

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Gene Construct

• Restriction enzyme digest of DNA fragment and
  plasmid DNA
• Insertion of DNA fragment into vector
• Cohesive Ends
• Decameric Linker
• DNA Ligase

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Cloning Using Vectors

• Vector
• Enhance the delivery of recombinant DNA into
  bacteria
• Select the bacteria containing the vector
• Plasmids
• Circular duplex DNA
• Occur naturally in some bacteria
• Bacteriophages
• Virus
• Circular single-stranded DNA

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Plasmids

• Genes for production of toxins, breakdown of
  natural products, inactivation of antibiotics
• Accessory chromosomes can replicate independently
  of host chromosomes
• pBR322
• Genes for tetracycline and ampicillin resistance
• Eco RI site doesnt deactivate genes
• HindIII, SalI, BamHI sites deactivate gene for
  tetracycline resistance
• Selection of bacteria containing construct

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Bacteriophage

• Lambda (?) Phage
• Lytic Pathway
• Viral functions fully expressed
• Lysogenic Pathway
• Insert in host genome and replicated along with
  host genome
• Can remain inactive for years
• Environmental changes can trigger expression of
  dormant viral DNA
• Foreign DNA replaces a segment of ? phage DNA
  without affecting its function

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Bacteriophage

• M13 Phage
• Useful for sequencing inserted DNA
• Doesnt kill host
• Single-stranded DNA
• Replicative form is double-stranded but
  single-strand package into viral particle
• Sequencing
• Single-stranded DNA produced
• DNA Polymerase

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Artificial Chromosomes

• Bacterial Artificial Chromosomes (BACs)
• Yeast Artificial Chromosomes (YACs)
• Autonomous replication sequence, a centromer, a
  pair of telomeres, selectable marker geners, and
  cloning site
• Restriction enzymes
• Separate fragments by pulsed-field gel
  electorphoresis
• Ligated to YACs

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Genomic Library

• Total genomic DNA
• Fragment genomic DNA
• Restriction enzymes
• Mechanically shearing
• Separate by size 15 kb
• Electrophoresis

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Genomic Library

• Gene Construct
• Synthetic Linkers added
• Fragments inserted into vector
• Cloning
• Infect bacteria with phage
• Phage Propagated indefinitely
• Genomic library
• Screening for gene of interest using DNA
  hybridization

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Complementary Probe

• mRNA from cells in which gene is available
• mRNA for hemoglobin in red blood cells
• Purify the mRNA
• cDNA synthesized in vitro
• cDNA cloned to produce probe
• Prepare from amino acid sequence of the protein
  encoded by the gene
• Obtain DNA sequence from amino acid sequence
• Met, Trp

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Designed Mutagensis

• Deletions
• Restriction digest of plasmid and ligate to form
  smaller circle
• Single cut, exonuclease treatment, and ligate to
  form circle

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Designed Mutagensis

• Substitutions
• Point Mutation
• Prepare prime with mutation use to synthesize DNA

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Designed Mutagensis

• Insertions
• Synthetic double-stranded oligonucleotide with
  desired mutations ligated into plasmid

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Designer Genes

• Create novels proteins
• Splicing different gene segments
• Synthetic gene via solid-phase method

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Manipulating Genes

• Bacteria can be used to produce a protein product
• Eukaryotic DNA has introns and exons
• Prokaryotic DNA does not have introns and exons

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Problem

• How can a prokaryotic system process eukaryotic
  DNA????

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Obstacle

• They Can Not!!!!!!!!!!!!!!!!

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Solution

• cDNA
• What is cDNA?

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cDNA

• Complementary DNA DNA is complementary to mRNA
• mRNA ? cDNA
• Reverse Transcriptase
• mRNA has poly (A) tail make a DNA primer
  containing oligo(T) residues
• RNA-DNA hybrid raise pH to hydrolyze RNA
• ssDNA ? dsDNA
• Terminal transferase adds dG to 3 end and oligo
  (dC) as primer

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Alkali Digestion of mRNA
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