DNA Technologies and Genomics

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DNA Technologies and Genomics

• Chapter 18

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Why It Matters

• Snowball Key to a Murder

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Biotechnology

• Biotechnology
• Any technique applied to biological systems to
  manipulate processes
• DNA technologies isolate purify, analyze and
  manipulate DNA sequences
• DNA fingerprinting used in forensics
• Genetic engineering uses DNA technologies to
  alter genes for practical purposes

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18.1 DNA Cloning

• Bacterial enzymes (restriction endonucleases)
  form the basis of DNA cloning
• Bacterial plasmids illustrate the use of
  restriction enzymes in cloning
• DNA libraries contain collections of cloned DNA
  fragments
• Polymerase chain reaction (PCR) amplifies DNA in
  vitro

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Recombinant DNA

• DNA cloning provides many copies of a gene
• Used for research or manipulation
• Recombinant DNA contains DNA from multiple
  sources joined together
• Recombinant plasmids containing gene of interest
  can be cloned in E. coli

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Cloning DNA Fragments
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Endonucleases

• Restriction enzymes (endunucleases) cut DNA at
  specific sequences in restriction sites
• Restriction fragments result
• Sticky ends have unpaired bases at cuts which
  will hydrogen bond
• Ligase stitches together paired sticky ends

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Restriction Enzyme EcoRI
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Plasmid Cloning Vectors

• Engineered to contain gene of interest and
  sorting genes
• Sorting genes identify E. coli with cloned
  plasmid
• E. coli with appropriate plasmid are ampicillin
  resistant and blue-white screened on X-gal

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Plasmid Cloning
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DNA Hybridization

• Uses nucleic acid probe to identify gene of
  interest in set of clones
• Probe has tag for detection
• Identified colony produces large quantities of
  cloned gene

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DNA Hybridization
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DNA Libraries

• Genomic libary
• Clones containing every sequence in a genome
• Used to isolate genes or DNA sequences
• Complementary DNA (cDNA) library
• DNA sequences made from expressed RNA
• mRNA extracted from cell
• Reverse transcriptase makes cDNA
• Removes introns for genetic engineering

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Polymerase Chain Reaction

• Polymerase chain reaction (PCR)
• Produces many sequence copies without host
  cloning
• Amplifies known DNA sequences for analysis
• Only copies sequence of interest
• Primers bracket sequence
• Agarose gel electrophoresis
• Separates fragments by size and charge
• Gel molecular sieve

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Polymerase Chain Reaction
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Agarose Gel Electrophoresis
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18.2 Application of DNA Technologies

• DNA technologies are used in molecular testing
  for many human genetic diseases
• DNA fingerprinting used to identify human
  individuals and individuals of other species
• Genetic engineering uses DNA technologies to
  alter the genes of a cell or organism
• DNA technologies and genetic engineering are a
  subject of public concern

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RFLPs

• Restriction fragment length polymorphisms
• DNA sequence length changes due to varying
  restriction sites from same region of genome
• Sickle cell anemia has RFLPs
• Southern blot analysis uses electrophoresis, blot
  transfer, and labeled probes to identify RFLPs
• Alternative is PCR and electrophoresis

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Sickle-Cell RFLPs
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Southern Blot Analysis
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DNA Fingerprinting

• Distinguishes between individuals
• Uses PCR at multiple loci within genome
• Each locus heterozygous or homzygous for short
  tandem repeats (STR)
• PCR amplifies DNA from STR
• Number of gel electrophoresis bands shows
  amplified STR alleles
• 13 loci commonly used in human DNA fingerprinting

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Forensics and Ancestry

• Forensics compares DNA fingerprint from sample to
  suspect or victim
• Usually reported as probability DNA came from
  random individual
• Common alleles between children and parents used
  in paternity tests
• Same principle used to determine evolutionary
  relationships between species

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DNA Fingerprint
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Genetic Engineering

• Transgenic organisms
• Modified to contain genes from external source
• Expression vector has promoter in plasmid for
  production of transgenic proteins in E. coli
• Example Insulin
• Protocols to reduce risk of escape

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Animal Genetic Engineering

• Transgenic animals used in research, correcting
  genetic disorders, and protein production
• Germ-line cell transgenes can be passed to
  offspring (somatic can not)
• Embryonic germ-line cells cultured in quantity,
  made into sperm or eggs
• Stem cells

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Transgenic Mice
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Genetically Engineered Mouse
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Gene Therapy

• Attempts to correct genetic disorders
• Germ-line gene therapy cant be used on humans
• Somatic gene therapy used in humans
• Mixed results in humans
• Successes for ADA and sickle-cell
• Deaths from immune response and leukemia-like
  conditions

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Animal Genetic Engineering

• Pharm animals produce proteins for humans
• Usually produced in milk for harmless extraction
• Cloned mammals produced by implantation of
  diploid cell fused with denucleated egg cell
• Low cloning success rate
• Increased health defects in clones
• Gene expression regulation abnormal

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Cloned Sheep

• Dolly

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Plant Genetic Engineering

• Has been highly successful
• Increased resistance to environmental effects and
  pathogens
• Plant pharms and increased nutrition
• Callus formation
• Ti (tumor inducing) plasmid from crown gall
  disease used as vector
• Transforming DNA (T DNA) genes expressed

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Crown Gall Tumor
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Ti Plasmid and Transgenic Plants
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Rhizobium radiobacter disarmed so cannot induce
tumors
Plant cell (not to scale)
Nucleus
T DNA with gene of interest integrated into plant
cell chromosome
Regenerated transgenic plant
Fig. 18-15b, p. 389
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GMO Concerns

• Genetically modifed organisms (GMOs) are
  transgenic and raise certain concerns
• Effect on environment
• Interbreeding with or harming natural species
• Cartagena Protocol on Biosafety provides rules on
  GMOs
• Stringent laboratory standards for transgenic
  organisms
• No bacterial escapes from labs

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GMO Tobacco
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GMO Rice
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18.3 Genome Analysis

• DNA sequencing techniques are based on DNA
  replication
• Structural genomics determines the complete DNA
  sequence of genomes
• Functional genomics focuses on the functions of
  genes and other parts of the genome

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18.3 (cont.)

• Studying the array of expressed proteins is the
  next level of genomic analysis
• Systems biology is the study of the interactions
  between all the components of an organism

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Genome Analysis

• Genomics
• Analyzes organization of complete genome and gene
  networks
• Human Genome Project took 13 years (2003)
• Revolutionizing biology and evolutionary
  understanding

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DNA Sequencing

• Used for small DNA sequences to genomes
• Dideoxy (Sanger) method of sequencing
• Dideoxyribonucleotides have H bound to 3 C
  instead of OH
• DNA polymerases place dideoxyribonucleotides in
  DNA, stops replication
• Polyacrylamide gel separates strands varying by
  one nucleotide

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Dideoxy (Sanger) Method
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Genomic Analyses (1)

• Structural genomics
• Sequence genomes to locate genes and funtional
  sequenes
• Functional genomics
• Studies functions of genes and other parts of
  genome

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Genomic Analyses (2)

• Whole-genome shotgun method
• Breaks genome into many DNA fragments
• Computers assemble genome based on overlapping
  sequences

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Whole-Genome Shotgun Sequencing
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Functional Genomics

• Bioinformatics
• Analysis of large data sets
• Uses biology, computer science, mathematics
• Identify open reading frames with start and stop
  codons, sophisticated algorithms for introns
• Sequence similarity searches
• Genomics revealed many unknown genes
• Many genes similar between evolutionarily distant
  organisms

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Human Genome

• 3.2 billion base pairs
• Between 20,000 and 25,000 genes
• About 100,000 proteins
• Due to alternative splicing and protein
  processing
• Protein coding only 2 of genome
• 24 introns
• 50 repeat sequences of no known function

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Genome Analysis

• Data mining
• Gene functions
• Genome organization
• Expression controls
• Comparative genomics (with other organisms)
• Tests evolutionary hypotheses

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DNA Microarrays

• DNA microarrays (chips)
• About 20 nucleotide-long DNA probe sequences
• cDNA probes made from isolated mRNA
• Probes red or green from different cell states
• cDNA from each cell state hybridize with
  complementary sequences on chip
• Used to determine how expression changes in
  normal and cancer cells
• Also used to detect mutations

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DNA Microarray Analysis
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Proteomics

• Proteome
• Complete set of proteins expressed by genome
• Larger than genome in eukaryotes
• Proteomics (study of proteome)
• Protein microarrays (chips) similar to DNA
  microarrays
• Use antibodies to bind to proteins

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Systems Biology

• Studies organisms as a whole
• Investigates networks of genes, proteins, and
  biochemistry
• Combines genomics and proteomics with response to
  environment
• Complex data analysis and computer models
  limiting factors