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DNA Technology
Problem:
A chromosome can be millions of base pairs long.
How do you isolate and study a single gene?
Scientists had to find out how to cut, paste, and
copy DNA.
Fragile X chromosome
Cutting and Pasting DNA
• 1962 – DNA cutting enzymes isolated from
E. coli = restriction endonucleases, aka
“molecular scissors”
- left “sticky ends”
• Arthur Kornberg identified the pasting
mechanism for DNA
- an enzyme called ligase
- made artificial viral DNA loops
Werner Arber
Arthur Kornberg
Made “life in a
test tube”
Restriction Enzymes
Gene Cloning
Current applications:
A gene that originated in one
organism can be used to give
another organism a new
metabolic capability.
Useful protein products can be
harvested in large quantities
from bacterial cultures.
Can also be used for basic
research on genes or proteins.
Molecular cloning – isolating a
defined sequence of DNA and
making multiple copies of it “in
vivo”.
Using Reverse Transcriptase to
Synthesize cDNA
• Reverse transcriptase – an RNA-dependent DNA
polymerase
• Encoded by retroviruses – copy viral RNA into DNA
prior to integration into host
•Transcribes both
ss RNA and ss DNA
(needs a primer)
•Degrades the RNA from
RNA-DNA hybrids.
•Used to copy RNA into
complementary DNAs
(cDNAs).
Making cDNA for a Eukaryotic Gene
Using Reverse Transcriptase
Cloning a Eukaryotic Gene in a
Bacterial Plasmid
A “shotgun” approach
X-gal is hydrolyzed by B-galactosidase
to yield a blue product.
If a plasmid has foreign DNA inserted
into its lac Z gene, then the colony of
cells producing it will be white.
Can synthesize a nucleic acid probe
-cDNA (complementary to the gene of
interest) labeled with a radioactive
isotope or a fluorescent tag.
Using a Nucleic Acid Probe to
Identify a Cloned Gene
Cloning a eukaryotic gene into a
prokaryotic cell: problems and solutions
• Differences in gene expression (ex: promoters and other
DNA control sequences).
- Solution: expression vector - contains a prokaryotic promoter just
before the site where the eukaryotic gene is inserted.
• Presence of long introns in most eukaryotic genes (bacterial
cells do not have RNA-splicing machinery).
- Solution: make artificial eukaryotic genes that lack introns.
-extract processed RNA from the eukaryotic nucleus (no introns)
-use reverse transcriptase to make cDNA transcripts of this RNA.
-cDNA is attached to vector DNA for replication inside a cell.
-vector provides a bacterial promoter and any other necessary
control elements
Using Eukaryotic Cells for Cloning:
Yeast
• Avoids eukaryotic-prokaryotic incompatibility
• Yeast – single-celled, easy to grow, have plasmids
• Scientists have constructed vectors called
yeast artificial chromosomes (YACs)
-have an origin for DNA replication, a centromere, and two
telomeres
-behave normally in mitosis – foreign DNA cloned as the yeast cell
divides
-can carry more DNA than a plasmid vector
• Problem: many eukaryotic proteins have to be modified
before they are functional (addition of carbohydrate or
lipid groups)
-Yeast cells may not be able to modify the protein correctly
Artificial Chromosomes
Mammalian satellite DNA-based
artificial chromosomes (SATACs)
Use of host cells from animal
or plant cell cultures
• Many kinds of eukaryotic cells can take up foreign DNA
– but not very efficiently
• Scientists have developed a variety of more aggressive
methods:
-electroporation – apply a brief electrical pulse to a solution
containing cells. Creates a temporary hole in the CM thru
which DNA can enter.
-inject DNA directly into cells with a microscopic needle
-in plants, can attach DNA to microscopic particles of metals
and fire the particles into cells with a “gene gun”
DNA Libraries
• DNA fragments cloned at random into a plasmid
vector - the majority of genetic information will be
included in the mixture of bacteria (“shotgun”
approach)
• Cultures of the bacteria, collectively contain all the
genes and are called a library.
cDNA Libraries
• Partial genomic library
• Produced using the mRNA molecules
isolated from a cell.
• Contains only the genes that are
expressed (transcribed) within the cell.
• Advantage – can study the genes
responsible for specialized functions in
specific cell types.
The Polymerase Chain Reaction
(PCR)
• A technique of quickly
amplifying DNA without using
cells
• DNA contains the sequence
“targeted” for copying
• A heat-resistant DNA
polymerase is added (isolated
from bacteria living in hot
springs!)
• Plus a supply of all four
nucleotides and primers
• Primers are short, synthetic
molecules of single-stranded
DNA complementary to the
ends of the targeted DNA
• Each cycle takes only about 5
minutes to complete
DNA Analysis and Genomics
• Genomics – sequencing and studying whole sets of genes and their
interactions - to make comparisons between cells, individuals, and
species.
• Gel electrophoresis – separates nucleic acids and proteins based
on size and charge
• Pure samples of these bands can be recovered from the gel and
retain their biological activity.
Restriction Fragment Analysis
by Southern Blotting
1. Restriction enzyme applied.
2. Gel separation.
3. Blot onto nitrocellulose paper.
4. Ss-DNA probes added.
5. Hybridization.
6. Bands identified ARG.
RFLP
Restriction Fragment Length
Polymorphisms (RFLPs)
• Used to find differences in noncoding
sequences of DNA.
• Can serve as genetic markers for a particular
location (locus) in the genome.
• A given RFLP marker frequently occurs in
numerous variants in a population (hence,
polymorphisms)
A DNA fingerprint is a specific
pattern of RFLP bands
STRs – variations in
number of tandem
repeated base
sequences found in
satellite DNA.
Human DNA Fingerprinting
Father and four children.
Which lane contains the
DNA of the father?
Lane 3 is the only lane
that shares one band
with each of the other lanes.
Which child is least
likely to be the biological
offspring of these
parents?
Child 2
The Human Genome Project
• Goals:
1. Determine nucleotide sequence for entire human
genome (3 X 109 bps).
2. Map the location of every gene on each chromosome.
3. Compare to genomes of other organisms.
• Fundamental questions:
1.
2.
3.
4.
How are genomes organized?
How is gene expression controlled?
How are cell growth and differentiation under genetic control?
How does evolution occur?
• Scientists have discovered specific genes responsible
for several genetic disorders:
1. Cystic fibrosis
2. Duchenne muscular dystrophy
3. Colon cancer
Human Genome Project
• International consortium of 20 groups of
researchers.
• Included mapping of important research organisms –
E. coli, yeast, C.elegans (nematode), and mouse.
• Three stages:
1. Genetic (linkage) mapping – develop a map of several
thousand genetic markers (genes, RFLPs, STRs)
2. Physical mapping: ordering DNA fragments using restriction
fragments (overlap)
3. DNA sequencing using clones of short DNA fragments and,
later, DNA sequencing machines.
• Later, J.C. Ventra (Celera Genomics) used powerful
computer programs to order the large number of
random short sequences cut from the whole genome.
Studying Gene Expression
Red – genes expressed in
sample A.
Green – genes expressed in
sample B.
Yellow – genes expressed
equally in both samples.
Used to determine which genes
are expressed in response to a
specific treatment or disease.
Also – tissue specific genes.
DNA microarray full set of 1,000s
of sequences
DNA Microarray Assays
Determining Gene Function
• In vitro mutagenesis:
1. Changes are made to a gene
2. Altered gene returned to cell
3. Monitor changes in physiology
or developmental patterns
• RNA interference (RNAi):
- Synthetic double-stranded RNA molecules that match a
gene sequence trigger the breakdown of that gene’s
mRNA.
Practical Applications of DNA
Technology
• Medicine:
-Diagnosis of infectious diseases and genetic disorders
-Human gene therapy – currently aimed at fighting heart
disease and cancer
-Pharmaceutical products – growth hormones, insulin,
vaccines
• Forensics:
-DNA fingerprints
-Use simple tandom repeats (STRs) found in satellite DNA
-Five small regions of the genome known to vary widely
Practical Applications of DNA
Technology (cont.)
• Environmental – genetically engineered
organisms:
– Able to extract heavy metals
– Sewage treatment
– Research – engineering organisms to degrade chlorinated
hydrocarbons and other toxic chemicals
- Bioremediation – cleaning up oil spills and waste dumps
• Agricultural:
- Transgenic organisms
-increased productivity
-pest resistance, disease resistance
-”pharm” animals
-”golden rice” – enriched with beta-carotene
Safety and Ethical Questions