CHAPTER 8: RECOMBINANT DNA TECHNOLOGY Restriction Enzymes

The Role of Recombinant DNA Technology in  Bacterial enzymes that cut DNA molecules only at
Biotechnology restriction sites
 Restriction site sequences are usually
 Biotechnology – the use of microorganisms to
palindromes
make practical products
 Categorized into two groups based on type of cut
 Recombinant DNA technology
 Cuts with sticky ends
 Intentionally modifying genomes of
 Cuts with blunt ends
organisms for practical purposes
 Three goals Vectors
 Eliminate undesirable phenotypic
traits  Nucleic acid molecules that deliver a gene into a
 Combine beneficial traits of two or cell
more organisms  Useful properties
 Create organisms that synthesize  Small enough to manipulate in a lab
products humans need  Survive inside cells
 Contain recognizable genetic marker
The Tools of Recombinant DNA Technology  Ensure genetic expression of gene
 Include viral genomes, transposons, and plasmids
Mutagens
Gene Libraries
 Physical and chemical agents that produce
mutations  A collection of bacterial or phage clones
 Scientists utilize mutagens to  Each clone in library often contains one gene
 Create changes in microbes' genomes to of an organism’s genome
change phenotypes  Library may contain all genes of a single
 Select for and culture cells with beneficial chromosome
characteristics  Library may contain set of cDNA complementary
 Mutated genes alone can be isolated to mRNA
The Use of Reverse Transcriptase to Synthesize cDNA Techniques of Recombinant DNA Technology
 Isolated from retroviruses Multiplying DNA in vitro: The Polymerase Chain Reaction
 Uses RNA template to transcribe molecule of (PCR)
cDNA
 Easier to isolate mRNA molecule for desired  Large number of identical molecules of DNA are
protein first produced in vitro
 cDNA generated from mRNA of eukaryotes has  Critical to amplify DNA in variety of situations
introns removed  Epidemiologists use to amplify genome of
 Allows prokaryotic cells to produce unknown pathogen
eukaryotic proteins  Amplified DNA from Bacillus anthracis spores
in 2001 to identify source of spores
Synthetic Nucleic Acids  Repetitive process consisting of three steps
 Denaturation
 Molecules of DNA and RNA produced in cell-free
 Priming
solutions
 Uses of synthetic nucleic acids  Extension
 Can be automated using a thermocycler
 Elucidating the genetic code
 Creating genes for specific proteins Selecting a Clone of Recombinant Cells
 Synthesizing DNA and RNA probes to locate
specific sequences of nucleotides  Must find clone containing DNA of interest
 Synthesizing antisense nucleic acid molecules  Probes are used

Separating DNA Molecules: Gel Electrophoresis and the

Southern Blot
  Gel electrophoresis  Locating genes on a nucleic acid molecule
 Separates molecules based on electrical  Provides useful facts concerning metabolism,
charge, size, and shape growth characteristics, and relatedness to others
 Allows scientists to isolate DNA of interest  Locating genes
 Negatively charged DNA drawn toward  Until 1970, genes were identified by labor-
positive electrode intensive methods
 Agarose makes up gel; acts as molecular sieve  Simpler and universal methods are now
 Smaller fragments migrate faster and farther available
than larger ones  Restriction fragmentation
 Determine size by comparing distance  Determine relative location of DNA
migrated to standards fragments produced by cleavage with
 Southern blot restriction enzymes
 DNA is transferred from gel to nitrocellulose  Fluorescent in situ hybridization (FISH)
membrane  Fluorescent probe used to visualize
 Probes are used to localize DNA sequence of location of a gene
interest  Nucleotide sequencing
 Northern blot – similar technique used to  Genomics – sequencing and analysis of the
detect RNA nucleotide bases of genomes
 Uses of Southern blots  Elucidation of the genomes of pathogens is a
 Genetic "fingerprinting" priority
 Diagnosing infectious disease  Used to relate DNA sequence to protein
 Demonstrating presence of organisms that function
cannot be cultured
Environmental Studies
DNA Microarrays
 Most microorganisms have never been grown in
 Consist of molecules of immobilized single- a laboratory
stranded DNA  Scientists know them only by their DNA
 Fluorescently labeled DNA washed over array will fingerprints
adhere only at locations where there are  Allowed identification of over 500 species of
complementary DNA sequences bacteria from human mouths
 Variety of scientific uses of DNA microarrays  Determined that methane-producing archaea
 Monitoring gene expression are a problem in rice agriculture
 Diagnosing infection
Pharmaceutical and Therapeutic Applications
 Identifying organisms in an environmental
sample  Protein synthesis
 Creation of synthetic proteins by bacteria and
Inserting DNA into Cells
yeast cells
 Goal of DNA technology is insertion of DNA into  Vaccines
cell  Production of safer vaccines
 Natural methods  Subunit vaccines
 Transformation  New approaches to stimulate immunological
 Transduction memory
 Conjugation  Introducing genes of pathogens into
 Artificial methods fruits and vegetables
 Electroporation  Injecting humans with plasmid carrying
 Protoplast fusion gene from pathogen
 Injection – gene gun and microinjection - Humans synthesize pathogen's
proteins

Applications of Recombinant DNA Technology  Genetic screening

Genetic Mapping
  DNA microarrays are used to screen  Enzyme that breaks down pectin is
individuals for inherited disease caused by suppressed in some tomatoes
mutations  Allows tomatoes to ripen on vine and
 Can also identify pathogen's DNA in blood or increases shelf life
tissues  BGH allows cattle to gain weight more rapidly
 DNA fingerprinting  Have meat with lower fat content and
 Identifying individuals or organisms by their produce 10% more milk
unique DNA sequence  Gene for β-carotene (vitamin A precursor)
 Gene therapy are inserted into rice
 Missing or defective genes are replaced with  Scientists considering transplanting genes
normal copies coding for entire metabolic pathways
 Some patients' immune systems react
The Ethics and Safety of Recombinant DNA Technology
negatively
 Medical diagnosis  Long-term effects of transgenic manipulations are
 Patient specimens can be examined for unknown
presence of gene sequences unique to  Unforeseen problems arise from every new
certain pathogens technology and procedure
 Xenotransplants  Natural genetic transfer could deliver genes from
 Animal cells, tissues, or organs introduced transgenic plants and animals into other
into human body organisms
Agricultural Applications  Transgenic organisms could trigger allergies or
cause harmless organisms to become pathogenic
 Production of transgenic organisms  Studies have not shown any risks to human
 Recombinant plants and animals altered by health or environment
addition of genes from other organisms  Standards are imposed on labs involved in
 Herbicide tolerance recombinant DNA technology
 Gene from Agrobacterium tumefaciens  Can create biological weapons using same
conveys resistance to glyphosate (Roundup) technology
 Farmers can kill weeds without killing  Ethical issues
crops  Routine screenings?
 Salt tolerance  Who should pay?
 Scientists have inserted a gene for salt  Genetic privacy rights?
tolerance into tomato and canola plants  Profits from genetically altered
 Transgenic plants survive, produce fruit, and organisms?
remove salt from soil  Required genetic screening?
 Freeze resistance  Forced correction of "genetic
 Crops sprayed with genetically modified abnormalities"?
bacteria can tolerate mild freezes
 Pest resistance
 Bt toxin
 Naturally occurring toxin harmful only to
insects
 Used by organic farmers to reduce insect
damage to crops
 Gene for Bt toxin is inserted into various crop
plants
 Genes for Phytophthora resistance are
inserted into potato crops

 Improvements in nutritional value and yield