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DNA Technologies and Genomics
Chapter 18
Why It Matters
 Snowball: Key to a Murder
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
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
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
Cloning DNA
Fragments
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
Restriction Enzyme EcoRI
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
Plasmid Cloning
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
DNA Hybridization
DNA Libraries
 Genomic libary
• Clones containing every sequence in a genome
• Used to isolate genes or DNA sequences
 Complementary DNA (cDNA) library
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DNA sequences made from expressed RNA
mRNA extracted from cell
Reverse transcriptase makes cDNA
Removes introns for genetic engineering
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
Polymerase Chain Reaction
Agarose Gel Electrophoresis
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
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
Sickle-Cell RFLPs
Southern Blot Analysis
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
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
DNA Fingerprint
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
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
Transgenic Mice
Genetically
Engineered Mouse
Gene Therapy
 Attempts to correct genetic disorders
• Germ-line gene therapy can’t 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
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
Cloned Sheep
 “Dolly”
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
Crown Gall Tumor
Ti Plasmid and
Transgenic Plants
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
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
GMO Tobacco
GMO Rice
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
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
Genome Analysis
 Genomics
• Analyzes organization of complete genome and
gene networks
 Human Genome Project took 13 years (2003)
• Revolutionizing biology and evolutionary
understanding
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
Dideoxy (Sanger)
Method
Genomic Analyses (1)
 Structural genomics
• Sequence genomes to locate genes and funtional
sequenes
 Functional genomics
• Studies functions of genes and other parts of
genome
Genomic Analyses (2)
 Whole-genome shotgun method
• Breaks genome into many DNA fragments
• Computers assemble genome based on
overlapping sequences
Whole-Genome
Shotgun Sequencing
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
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
Genome Analysis
 Data mining
• Gene functions
• Genome organization
• Expression controls
 Comparative genomics (with other organisms)
• Tests evolutionary hypotheses
DNA Microarrays
 DNA microarrays (chips)
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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
DNA Microarray Analysis
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
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