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AP Biology Chapter 20
DNA Technology
DNA Technology
• Recombinant DNA: genes from 2 different
sources, can be 2 dif. species
• Genetic engineering: direct manipulation of
genes for practical purposes
• Biotechnology: manipulation of organisms or
their components for practical tasks (ancient
idea)
• Gene cloning vs. cell cloning and cloning of
whole organisms – including mammals
– Lots of copies of a gene to be used in research
– Cloning of bacteria, mice, pigs, etc for their products
– Making new organisms…..?
Bacterial Tools
Restriction Enzymes
• 1960’s
• Cut foreign DNA
• Recognize specific
sequences
• Leave sticky ends
• Cuts are reproducible
because they cut at
specific sequences
• Fusions occur with DNA
ligase
• Plasmids
• Multiple copies
– “clones” of the gene
• Genes that are frequently
expressed
• Can be transferred to
other cells
• DNA is universal, so
bacteria will produce
plasmids even if DNA is
not theirs originally
• Used as a “vector”
Problems and Solutions
• Expression vectors are used to get prok promoter “ON”
– Make cure inserted gene is ‘downstream’ from this type of
promoter
• Must remove introns for prok to read mRNA
– Start with mRNA from cells that have already created the edited
version and make something called cDNA (complementary)
• Use cells that aren’t bacteria/prok
– Yeast and single celled fungi and plants
– Also helps with modification that occurs to proteins after
translation in euk cells
• Use artificial chromosomes
– Origin, centromere and telomeres with foreign DNA
• Force uptake
– Euk cells exposed to pulses of current can allow DNA through
plasma membrane….
• Inject DNA
– Using “shot gun approach”
Genomic Libraries
• Genomic library is a collection of all the
recombinant plasmids in a particular
colony of bacteria
• cDNA libraries contain all the gene that
there are cDNA for
Polymerase chain reactions, etc.
• Gel electrophoresis employs electricity to separate
strands of DNA based on their size
• Bands of DNA can be identified using Southern blotting –
a small sequence is radioactively tagged, then put with
DNA and every where it pairs up with complementary
bases it can be read
• Restriction fragment length polymorphisms (RFLP’s)
– Differences in Southern blot bands between homologous
chromosomes, especially in noncoding areas of genome
• PCR is a technology that amplifies the DNA so there are
billions of copies
– DNA from woolly mammoth
– DNA from embryonic cells
– DNA from crime scenes for DNA fingerprinting
Diagrams
• Be sure to read information on the
following:
– Gel electrophoresis pg. 374
– RFLP’s and Southern Blotting pg. 375
– Sanger method of sequencing pg. 378
– Your lab manual – Lab 6 A and 6B
Human Genome Project
• Computer systems worked internationally for almost 15
years to list all the human genes, including the
noncoding regions.
• 3 billion base pairs long
• 100,000 genes
• Also mapped for research and comparison
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E. coli
(and other bacteria)
Saccharomyces cerevisiae
(yeast)
Caenorhabditis elegans (nematode/worm)
Drosophilia melanogaster (fruit fly)
Mus musculus
(mouse)
Danio rerio
(zebra fish)
Terms
• Genetic mapping: linkage map determined by recombination
frequencies
– Currently have 500 markers on human genome
• Physical mapping: map units
• DNA sequencing: list of bases for all 3million nucleotides pairs
• Genome analysis
– find genes by looking for sequences, that when transcribed have
a start and stop codon
– Compare sequences found in one organism and look for similar
sequence in other organsims
• Microarray assay: microscope slide with known genes in wells –
mRNA from a cell is obtained, reacted with cDNA, if bases match
they will pair up and when hybrid DNA is placed on slide it will glow
in the well if its for the gene in that well
• Genomics: the study of genomes and genes
Human Health Applications
1. Diagnosis of disease by showing a marker for
the gene
2. Human Gene Therapy when there is a known
allele
– Enhance immune system
– Fight cancer
– Stem cell products
3. Pharmaceuticals
– Manufacture proteins that are required
– Design vaccines that are specific and harmless
Environment, Agriculture
and Forensics
• Forensics uses RFLP, southern blot and gel
electrophoresis to create a DNA fingerprint
• Genetically engineered microbes can ‘feast’ on
oil spills and heavy metal contaminants in water
and soil
• Transgenic organisms are being developed as a
source of organs and as a source of medically
useful proteins
• Genetically engineered plants are created to
have specific useful features, as well as being
pest resistant and potentially able take up N
without fertilizers
Safety and Ethics
• Currently regulated by the following US
agencies
– USDA, United States Department of
Agriculture
– FDA, Food and Drug Administration
– EPA, Environmental Protection Agency
– NIH – National Institute of Health
Recombinant DNA Advisory Committee
– And potentially , voters like you….