Transcript LEQ: How do we splice new genes into DNA?

LEQ: HOW DO WE
SPLICE NEW GENES
INTO DNA?
12.1 to 12.7 and 12.18
RECOMBINANT DNA TECHNOLOGY
 This is a set of lab techniques for combining genes from
different sources – even different species – into a single
DNA molecule
 Began with the study of Eschericia coli (E. coli)
TERMS FOR RECOMBINANT DNA
TECHNOLOGY
 Gene Cloning – the production of
multiple copies of a gene
 Genetic Engineering – the direct
manipulation of genes for particle
purposes
 Biotechnology – the use of living
organism (often microbes) to
perform useful tasks
RESTRICTION ENZYMES
 Bacterial enzymes that cut foreign DNA; original purpose was a defense
mechanism of bacterial to protect against foreign DNA (phage DNA)
 Restriction enzymes are now used to cut DNA molecules in
reproducible ways
 These enzymes produce 2 different kinds of ends
1. Blunt ends – these enzymes cut straight through the double strand
of DNA; produces restriction fragments with no overlapping single
strands; fragments cannot bond with other pieces of DNA
2. Sticky ends – these enzymes have a staggered cut resulting in
fragments with single stranded ends which can bond with
complementary DNA
STICKY OR BLUNT?
Sticky
Blunt
Plasmids
small circular ring of DNA found
in prokaryotes and yeast
Important sites of plasmid
1. origin of replication
2. genetic marker (i.e.
antibiotic resistance)
3. restriction enzyme
cut sites
Restriction Enzymes &
Recombinant DNA
Restriction enzymes have
specific recognition sites
Scientists use a specific enzyme
to cut out a specific gene and a
specific plasmid
The sticky ends of the gene and
the plasmid will compliment
each other allowing scientists to
join the two
Use DNA Ligase to join two
DNA types to produce
recombinant DNA
Cloning Recombinant DNA
1.
Identify a restriction enzyme that will cut out gene of
interest and cut open the plasmid
2.
Isolate DNA from 2 sources (making sure the at the
plasmid has a genetic marker)
3.
Cut both types of DNA with the same restriction enzyme
4.
Mix the 2 types of DNA to join them through
complementary base pairing Add
5.
DNA ligase to bond DNA covalently producing
Recombinant DNA
6.
Incubate bacteria at 42 C with calcium chloride; bacteria
become competent / permeable - so that the bacteria
will take in the plasmid (TRANSFORMATION)
7.
Use a genetic marker to identify bacteria with the
recombinant plasmid
8.
Clone bacteria
Clone Storage via
Genomic Library
Plasmid Libraries and Phage
Libraries are created by cutting a
genome into fragments
Genome fragments are used to
create recombinant DNA
Recombinant DNA is then
either stored in a bacterial
culture or a phage culture
Reverse Transcriptase used
to make genes for cloning
1. Transcribe DNA into RNA in the
2.
3.
4.
5.
nucleus
RNA splicing occurs – removing introns
Isolate mRNA from the cell and add
reverse transcriptase to synthesize a
new strand of DNA
The mRNA is digested
Synthesize 2nd complimentary strand
using DNA polymerase creating
“cDNA”
What’s the difference?
DNA
 Contains introns; - must be
edited to be get to the DNA
that codes for a gene
cDNA
 No introns – the actual DNA
that codes for a particular gene
Genetically Modified
Organisms
Genetically modified organism –
an organism that acquires one
or more genes by artificial
means (gene may or may not be
from a different species)
Transgenic organism – organism
that contains a gene from
another species
Recombinant DNA
Applications
Bacteria are protein factories. E. coli is
the primary bacteria used; cheap and
easy to maintain cultures and
produce proteins (pharmaceutical
factories)
Products:
Chymosin (used for cheese
production), Human Insulin; Human
Growth Hormone; Factor VIII;
Hepatitis B vaccine; Diagnosis of HIV
Recombinant DNA
Applications
The yeast Saccharomyces
cerevisiae is commonly used
when eukaryotic cells are
needed.
Yeast serve as a good vector for
human genomic libraries
Recombinant DNA
Applications
Plants
By genetically modifying the genome
of a plant scientists have been able to
increase the nutritional value of
crops (i.e. “Golden Rice” contains
daffodil gene allowing it to produce
beta-carotene); scientists have been
able to genetically modify plants that
are drought resistant, pesticide
resistant, larger in size, and that have
a longer shelf life.
Recombinant DNA
Applications
Mammals
Recombinant DNA technology is used to add a
human gene for a desired human trait (protein)
to the genome of a mammal in such a way that
the gene’s products, such as antithrombin
(protein that prevents blood clots), are secreted
in the milk of the animal; Transgenic mammals
allow scientists to model human diseases and
find treatments to the diseases; Transgenic pigs
may serve as human blood and organ donors;
Transgenic cattle & fish have been engineered to
be larger in size – providing more meat
DNA Technology – pharmaceuticals and
Medicine
 Therapeutic Hormones –
human insulin produced by
bacteria (no longer use insulin
from cadavers or pigs)
 Diagnosis & Treatment of
disease – identify disease
causing alleles & tailored
treatments
 Vaccines – create harmless
variants of pathogen to
stimulate the immune system