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DNA Technologies and Genomics

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DNA Technologies and Genomics Chapter 18 – PowerPoint PPT presentation

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Title: DNA Technologies and Genomics


1
DNA Technologies and Genomics
  • Chapter 18

2
Why It Matters
  • Snowball Key to a Murder

3
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

4
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

5
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

6
Cloning DNA Fragments
7
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

8
Restriction Enzyme EcoRI
9
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

10
Plasmid Cloning
11
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

12
DNA Hybridization
13
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

14
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

15
Polymerase Chain Reaction
16
Agarose Gel Electrophoresis
17
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

18
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

19
Sickle-Cell RFLPs
20
Southern Blot Analysis
21
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

22
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

23
DNA Fingerprint
24
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

25
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

26
Transgenic Mice
27
Genetically Engineered Mouse
28
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

29
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

30
Cloned Sheep
  • Dolly

31
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

32
Crown Gall Tumor
33
Ti Plasmid and Transgenic Plants
34

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
35
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

36
GMO Tobacco
37
GMO Rice
38
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

39
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

40
Genome Analysis
  • Genomics
  • Analyzes organization of complete genome and gene
    networks
  • Human Genome Project took 13 years (2003)
  • Revolutionizing biology and evolutionary
    understanding

41
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

42
Dideoxy (Sanger) Method
43
Genomic Analyses (1)
  • Structural genomics
  • Sequence genomes to locate genes and funtional
    sequenes
  • Functional genomics
  • Studies functions of genes and other parts of
    genome

44
Genomic Analyses (2)
  • Whole-genome shotgun method
  • Breaks genome into many DNA fragments
  • Computers assemble genome based on overlapping
    sequences

45
Whole-Genome Shotgun Sequencing
46
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

47
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

48
Genome Analysis
  • Data mining
  • Gene functions
  • Genome organization
  • Expression controls
  • Comparative genomics (with other organisms)
  • Tests evolutionary hypotheses

49
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

50
DNA Microarray Analysis
51
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

52
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
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