DNA Technology
Genetic Engineering
Genetic engineering is the direct manipulation of genes for practical purposes Manufacturing protein products
Biotechnology is the manipulation of organisms or their components to make useful products Selective breeding of cattle Making wine and cheese
Genetic engineering/biotechnology are major areas of research in biology today
DNA Cloning Preparing identical copies of
a specific gene Can be used to produce a
protein product (gene protein)
Copies of a gene can be used for: Basic research Endowing an organism with
a new metabolic capability• pGLO
Practical Applications of Gene Cloning Gene for pest
resistance inserted into plants
Genes used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
Others??
Restriction Enzymes & Recombinant DNA Restriction Enzymes –
enzymes that cut DNA molecules at specific locations
Look for specific sequences:• Ie: ATTACCGTA
Recombinant DNA – DNA in which nucleotide sequences from 2
different sources (often different species) are combined into the same DNA molecule
Recombinant DNA
Recombinant DNA Sticky Ends:
Single-stranded ends of a restriction fragment (piece of DNA)
Sticky ends of restriction fragments can form hydrogen-bonded base pairs with other sticky ends These bonds are sealed
with DNA ligase
Plasmids
A plasmid is a DNA molecule that is not part of the chromosome in bacteria Often give bacteria an
“advantage” such as antibiotic resistance
Plasmids are often used as cloning vectors Take foreign DNA into a
cell and replicate there
Polymerase Chain Reaction (PCR)
PCR is a process used to copy a segment of one or many DNA molecules quickly in vitro (in a test tube)
PCR can make BILLIONS of copies of a target segment of DNA in a few hours
Polymerase Chain Reaction (PCR)
1. Denaturation Mixture is heated to separate the
DNA strands
2. Annealing Mixture is cooled to allow primers to
form (beginning of DNA replication)
3. Extension DNA polymerase adds nucleotides
to 3’ end of primer
Polymerase Chain Reaction (PCR)
Applications of Polymerase Chain Reaction (PCR) Used to amplify (copy) DNA from a variety
of sources Fragments of DNA from a 40,000-year-old
woolly mammoth DNA from fingerprints or tiny amounts of
blood, tissue, semen at crime scenes… If there is not much DNA available for
analysis, PCR can produce enough DNA for other applications (electrophoresis, etc.)
Restriction Fragment Analysis
Restriction fragment analysis detects certain differences in the nucleotide sequences of DNA molecules
Restriction Fragment Length Polymorphisms (RFLPs)
RFLPs are differences in restriction sites on homologous chromosomes that result in different restriction fragment patternsEveryone has a different DNA sequence,
so the restriction enzymes will cut the DNA at different places
The length of the restriction fragments can be analyzed using electrophoresis
Gel Electrophoresis
The DNA fragments produced by restriction enzymes are sorted by gel electrophoresis Sorted by rate of movement through a gel in
an electric field• Distance is inversely proportional to the length of
the fragment –• Long fragments don’t go far, short ones do
The banding pattern (where the fragments stop) is used to analyze/identify DNA
Gel Electrophoresis
The Human Genome Project Largely completed in
2003 Essentially, a detailed
map of the entire human genome Genetic mapping –
• Relative order of genes (1st, 2nd, 3rd, etc.)
Physical mapping –• Definitive distance
between genes (2457 bp, etc.)
DNA sequencing – • complete nucleotide
sequence (ATCCG…)
Applications of DNA Technology
Medical Applications Diagnosing genetic diseases
• Amplifying (cloning) genes and analyzing them
Gene therapy• Altering the genes of an
afflicted individual• Replacing a “defective” allele
with a “normal” allele• To be effective, it must be
done in cells that multiply throughout life
• Bone marrow cells (somatic cells)
• Gametic cells (sperm/egg)
• Lots of ethical issues involved here!
Applications of DNA Technology
Pharmaceutical ProductsUsing DNA cloning, genes that code
for the production of specific proteins can be made in vitro
• Human insulin (diabetes)• Human growth hormone (HGH)• Vaccines
Forensic Evidence
DNA fingerprinting (RFLP analysis and electrophoresis) is used to solve crimes The likelihood that 2 people
would have the same DNA fingerprint (restriction enzymes would cut their DNA at all the exact same places and therefore have the same banding pattern) is between 1 in 100,000 and 1 in 1,000,000,000
Also used in paternity testing
Applications of DNA Technology
Environmental CleanupMany bacteria can extract heavy
metals (copper, lead, nickel) from their environments
These genes could be added to other organisms to clean up mining wastes
Bacterial strains can degrade some of the chemicals released during oil spills
Ethics & DNA Technology
The progress in genetic technology has raised new ethical questions:Who should have the right to examine
someone else’s genes?How should that information be used?Should a person’s genome be a factor
in suitability for a job or eligibility for insurance?