Download Chapter 20 Notes: DNA Technology

Chapter 20 Notes:
DNA Technology
Understanding & Manipulating Genomes
• 1995: sequencing of the first complete genome
(bacteria)
• 2003: sequencing of the Human Genome mostly
completed
• These accomplishments depended on new technology:
– Recombinant DNA: DNA from 2 sources (often 2 species)
are combined in vitro into the same DNA molecule
• Called Genetic engineering: direct manipulation of genes for
practical purposes
 DNA technology has launched
a revolution in the area of:
 BIOTECHNOLOGY: the
use of living organisms or their
components to do practical
tasks
-microorganisms to make
wine/cheese
-selective breeding of
livestock
-production of antibiotics
-agriculture
-criminal law
**Practical goal of biotech =
improvement of human health
and food production
Ch 20 looks at:
1. Main techniques for manipulating DNA
2. How genomes are analyzed & compared
at the DNA level
3. Practical applications of DNA technology
(including social & ethical issues)
“Toolkit” for DNA technology involves:
-DNA vectors
-host organisms
- restriction enzymes
VECTORS = carriers for moving DNA
from test tubes back into cells
-bacterial plasmids (small, circular
DNA molecules that replicate within
bacterial cells)
-viruses
HOST ORGANISMS:
bacteria are commonly
used as hosts in genetic
engineering because:
1) DNA can easily be isolated from
& reintroduced into bacterial cells;
2) bacterial cultures grow quickly,
rapidly replicating any foreign
genes they carry.
RESTRICTION ENZYMES = enzymes that
recognize and cut short, specific nucleotide
sequences (called restriction sites)
-in nature, these enzymes protect the
bacterial cell from other organisms by
cutting up their foreign DNA
Restriction Enzymes (cont.)…
most restriction sequences are symmetrical
in that the same sequence of 4-8
nucleotides is found on both strands, but
run in opposite directions
restriction enzymes usually cut
phosphodiester bonds of both strands in a
staggered manner producing single
stranded “sticky ends”
Restriction Enzymes (cont.)…
“sticky ends” of restriction
fragments are used in the
lab to join DNA pieces
from different sources
(complementary base
pairing)
*RECOMBINANT DNA
unions of different DNA
sources can be made
permanent by adding DNA
ligase enzyme (form
covalent bonds between
bases)
DNA Technologies:
1) Cloning
2) DNA fingerprinting
(profiling)
3) Microarray
4) Gene therapy
Steps Involved in
Cloning a Human Gene:
Human gene
plasmid
1) Isolate human gene to clone (ex: insulin);
2) Isolate plasmid from bacterial cell;
3) cut both DNA samples with the same
restriction enzyme to open up bacterial
plasmid & create sticky ends on both
samples;
4) Mix the cut plasmids and human DNA genes
& seal with DNA ligase;
Cloning a Human Gene (cont.)…
5) Insert recombinant DNA plasmid back into
bacterial cell;
6) As bacterial cell reproduces, it makes copies
of the desired gene;
-grow cells on a petri dish
7) Identify cell clones carrying the gene of
interest.
-HOW? Which ones took up the gene & are
making insulin?
*Add a 2nd gene besides insulin; add one for antibiotic
resistance & then grow bacteria on that antibiotic
LE 20-4_3
Bacterial cell
Isolate plasmid DNA
and human DNA.
lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Cut both DNA samples with
the same restriction enzyme.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X-gal.
Incubate until colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid with
disrupted lacZ gene
Bacterial
clone
Why can bacteria produce insulin through recombinant
DNA technology? The genetic code is universal!!!!