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DNA Technology
• Recombinant DNA technology– Set of techniques for recombining
genes from different sources in vitro and transferring the recombinant DNA to a cell where it is expressed
– Typically uses a plasmid as its vector
– Same restriction enzymes used to make “sticky end” cuts
Restriction Enzymes
• Cut DNA after specific base sequences = restriction site
• Protect bacteria– cut up foreign DNA
• Sticky ends– jagged cut so other DNA can join
• DNA ligase– makes addition of DNA permanent
DNA Technology
• Biotechnology– Refers to the use of living organisms
or components to do practical tasks– Wine industry– Cheese industry– Selective breeding of livestock and
crops– Production of antibiotics
Cloning a Eukaryotic Gene
• Isolation of vector & gene-source DNA– Cloning vector is the original plasmid
• Insertion of DNA into vector– Use of restriction enzymes– May need to make cell competent (E. coli)
• Introduction of cloning vector into cells– Naked DNA added to culture– Bacteria take in plasmid by
transformation
Cloning a Eukaryotic Gene
• Cells reproduce in a culture– Transformed cells are producing new
cells with the cloned gene
• Identification of cell clones– Typically use the R plasmid for ampicillin
resistance– Only cells that have transformed can
grow on the ampicillin agar– Nucleic acid hybridization
Polymerase Chain Reaction• Use when source of DNA is impure or
scarce• Clones DNA entirely in vitro• Making many copies of a specific segment
of DNA (billions of copies in a few hours)• Used for DNA analysis
– Ancient DNA fragments– DNA from tiny samples– DNA from single embryonic cells– DNA of viral genes
Polymerase Chain Reaction
• Devised in 1985
• Starting materials:– DNA polymerase, primers, nucleotides
Polymerase Chain Reaction
• Heat to separate DNA strands– use DNA polymerase from a bacteria
that lives in hot springs
• Cool to allow primers to bind
• DNA polymerase extends the 3’ end of each primer
• Multiplies exponentially
Southern Blots• Hybridization technique that enables
researchers to determine the presence of certain nucleotide sequences in a sample of DNA
• RFLP’s– differences in DNA sequence on homologous
chromosomes that result in different patterns of restriction fragment lengths for every species
– useful as genetic markers– Inherited following Mendel’s patterns
Southern Blots
• Combination of 5 techniques– Restriction fragment preparation
– Electrophoresis
– Blotting (DNA bands transferred to nitrocellulose paper)
– Hybridization with radioactive probe (attach to gene of interest)
– Autoradiography
Practical Applications• Diagnosis
– early detection of disease before symptoms show or even birth
– use probes with cloned genes
• Human gene therapy– traceable genetic disorders– may eventually be correctable– replace defective genes with functional genes– only effective if cells receiving normal allele
rapidly reproduce
Practical Applications
• Environmental– microorganisms to get rid of waste
– mining
– recycling of wastes and detoxifying
– sewage treatment plants
Practical Applications• Pharmaceutical products
– insulin, growth hormone
• Forensics– blood and tissue type
– RFLP and Southern Blots
• Agricultural– animal husbandry
– cellulase