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A. Chapter 20~
DNA
Technology
&
Genomics
I. Intro: Vocabulary
A. Recombinant DNA: DNA in
which genes from 2
different sources are
combined
B. Genetic engineering: direct
manipulation of genes for
practical purposes
C. Biotechnology: manipulation
of organisms or their
components to perform
practical tasks or provide
useful products
Overview: Use of Bacterial Plasmids
in Gene Cloning
II. DNA Cloning
A. Cloned genes used for basic research
and commercial products
1. A foreign gene is inserted into a bacterial
plasmid and this recombinant DNA molecule
is returned to a bacterial cell.
2. Every time this cell reproduces, the
recombinant plasmid is replicated as well
and passed on to its descendents.
3. Under suitable conditions, the bacterial
clone will make the protein encoded by the
foreign gene.
B. Restriction
enzymes
(endonucleases):
in nature, these
enzymes protect
bacteria from
intruding DNA
1. they cut up the
DNA (restriction)
2. very specific
C. Restriction site:
recognition
sequence for a
particular
restriction enzyme
D. Restriction fragments: segments of
DNA cut by restriction enzymes in a
reproducable way
E. Sticky end: cut covalent
phosphodiester bonds of both strands
= short extensions of restriction
fragments
F. DNA ligase: enzyme that can join the
sticky ends of DNA fragments
G. Cloning vector: DNA molecule that
can carry foreign DNA into a cell and
replicate there (usually bacterial
plasmids)
III. Genes can be cloned in
DNA vectors
A. Recombinant plasmids- splicing restriction
fragments from foreign DNA into plasmid
1. can be returned relatively easily to bacteria
2. cloning vector- a DNA molecule that can carry
foreign DNA into a cell and replicate there
B. As a bacterium carrying a recombinant plasmid
reproduces, the plasmid replicates within it
C. Bacteria are most commonly used as host
cells:
1. DNA can be easily isolated
2. Then, reintroduced into their cells
3. the cultures grow (replicate) quickly
D. Steps for
eukaryotic
gene
cloning
1. Isolation of cloning vector (bacterial
plasmid) & gene-source DNA (gene of
interest)
2. Insertion of gene-source DNA into the
cloning vector using the same restriction
enzyme; bind the fragmented DNA with
DNA ligase
3. Introduction of cloning vector into cells
(transformation by bacterial cells)
4. Cloning of cells (and foreign genes)
5. Identification of cell clones carrying the
gene of interest, one way is nucleic acid
hybridization using a nucleic acid probe
Plasmid Cloning
http://www.sumanasinc
.com/webcontent/anisa
mples/molecularbiolog
y/plasmidcloning_fla.ht
ml
How do we know which
colonies contain the
cloned gene?
-Look for the gene or
its protein product
– After
denaturation
(separating) the
DNA strands in
the plasmid, the
probe will
hydrogen-bond to
its
complementary
sequence,
tagging colonies
with the targeted
gene
F. Solutions to Problems
Expressing Eukaryotic Genes
1. Use expression vector, a cloning vector
containing the prokaryotic promotor
upstream of the restriction site
1. bacterial host recognizes the promotor and
expresses the foreign gene
2. Introns in eukaryotic genes
1. A processed mRNA acts as the template for
making a complementary DNA (cDNA) by
reverse transcription. cDNA, with a promoter,
can be attached to a vector for replication,
transcription, and translation inside bacteria.
cDNA
3. Use eukaryotic cells as host for genes
1. Yeast cells, single-celled fungi, are as easy to
grow as bacteria and have plasmids, (rare for
eukaryotes)
2. Scientists have constructed yeast artificial
chromosomes (YACs) - an origin site for
replication, a centromere, and two telomeres
3. carry more DNA than a plasmid
4. Host provides the modifications after
translation needed by many proteins
A. includes adding carbohydrates or lipids
4. When DNA not taken up efficiently
1. electroporation, brief electrical pulses create
a temporary hole in plasma membrane
2. Or, scientists can inject DNA into cells using
microscopically thin needle
G. Polymerase chain reaction (PCR)
1. Devised in 1985
2. Quick amplification of any piece of
DNA without cells (in vitro)
3. PCR can make billions of copies of a
targeted DNA segment in a few
hours
A. three-step cycle: heating, cooling,
and replication
B. Applications: fossils (40,000 yr old
wooly mammoth), forensics,
prenatal diagnosis, etc.
http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
DNA
incubated in
test tube w/:
-special DNA
polymerase
-supply of
nucleotides
-short pieces
of DNA primer
IV. DNA Analysis & Genomics
We have gene segments,
now what?
Genomics- Comparisons among
whole sets of genes & interactions
A. Gel electrophoresis
B. Restriction fragment
analysis (RFLPs)
C. Southern blotting
D. DNA sequencing
E. Human genome
project
A. Gel Electropheresis
A. Gel electrophoresis: separates nucleic
acids or proteins on the basis of size or
electrical charge creating DNA bands of
the same length
1. DNA molecule separation depends mainly on
size (length of fragment) with longer
fragments migrating less along the gel
Gel Electropheresis
http://www.sumanasinc.com/webco
ntent/anisamples/majorsbiology/
gelelectrophoresis.html
B. Restriction fragment analysis
1.
Separated fragments can
be recovered undamaged
from gels, providing pure
samples of individual
fragments.
Distinguish different alleles
(specific to one base pair)
2. Although electrophoresis will yield too
many bands to distinguish individually,
we can use nucleic acid hybridization
with a specific probe to label discrete
bands that derive from our gene of
interest.
3. The radioactive label on the singlestranded probe can be detected by
autoradiography
4. restriction fragment length
polymorphisms (RFLPs) can serve
as genetic markers for a particular
location (locus) in the genome
C. Southern Blotting
1. (Southern hybridization) the transfer
of the DNA fragments from the gel to
a sheet of nitrocellulose paper
A. Fragments separated by size
B. denatures the DNA fragments
2. Bathe sheet in solution containing a
probe
A. probe attaches by base-pairing (hybridize)
to the DNA sequence of interest
3. Visualize bands containing the label
with autoradiography
Three individuals, the results of these steps
show that individual III has a different
restriction pattern than individuals I or II.
D. Entire genomes can be
mapped at the DNA level
1. Human Genome Project, begun in
1990
A. RFLPs serve as the basis of a detailed map
of the entire human genome
2. Other organisms important to biological
research with entire genomes mapped:
E. coli, yeast, fruit fly, and mouse
3. Three phases to sequencing:
1. genetic (linkage) mapping
2. physical mapping
3. DNA sequencing
4. Genetic mapping- use linkage
maps to locate genetic markers
throughout chromosomes
1. Based on recombination frequencies
2. Markers may be known segements
of DNA, RFLPs, and microsatellites
5. Physical mapping- determining
order of identified restriction
fragments
1. Chromosome walking- using known
segments to make a map of
overlapping fragments
Chromosome
walking
6. DNA sequencing- the long fragments
are then cut, cloned and sequenced
7. Sanger Method- deriving the sequence
in a method similar to PCR
1. Special dideoxynucleotides used in
reaction, do not copy the whole template,
instead, fragments of various lengths
2. dideoxynucleotides, marked radioactively
or fluorescently, lack a 3’-OH to attach the
next nucleotide
8. The order of these fragments via gel
electrophoresis can be interpreted as
the nucleotide sequence
J. Craig Venter (Celera Genomics) decided
in 1992 to try a whole-genome shotgun
approach
9. The progess
A. In 1995, Venter announced genome of a
bacterium
B. In 2000, he finished Drosophila
melanogaster
C. In February, 2001, Celera and the public
consortium separately announced
sequencing over 90% of the human
genome
D. By mid-2001, the genomes of about 50
species had been completely (or almost
completely) sequenced
E. There are still gaps in the human
sequence
enormous amounts of noncoding DNA
E. Evolutionary Significance
1. Comparisons of genome sequences
confirm very strongly the evolutionary
connections between even distantly
related organisms and the relevance of
research on simpler organisms to our
understanding of human biology.
A. yeast genes can substitute for human versions
B. Understand human disease gene by studying
its normal counterpart in yeast
C. Bacterial sequences reveal unsuspected
metabolic pathways that may have industrial
or medical uses
F. Unknown gene functions
1. disable the gene and hope that the
consequences provide clues to the gene’s
normal function
A. Using in vitro mutagenesis, specific
changes are introduced into a cloned gene,
altering or destroying its function.
B. When the mutated gene is returned to the
cell, it may be possible to determine the
function of the normal gene by examining
the phenotype of the mutant.
2. In nonmammalian organisms, a simpler
and faster method, RNA interference
(RNAi), has been applied to silence the
expression of selected genes.
What’s Next?
A. The next step is proteomics, the
systematic study of full protein sets
(proteomes) encoded by genomes.
B. Challenges:
1. The sheer number of proteins in humans
due to:
A. alternative RNA splicing
B. post-translational modifications
2. Collecting proteins because a cell’s
proteins differ with cell type and its state
3. Proteins are extremely varied in structure
and chemical and physical properties
V. Practical DNA Technology
Uses
A. Diagnosis of disease
B. Human gene therapy
C. Pharmaceutical products
(vaccines)
D. Forensics
E. Animal husbandry (transgenic
organisms)
F. Genetic engineering in plants
G. Ethical concerns?
Human Gene
Therapy
DNA fingerprints can be used
forensically to presence evidence to
juries in murder trials.
the blood on the clothes is from the victim,
not the defendant.
Agricultural Use
A. Crop plants with genes for desirable traits
1. delayed ripening and resistance to spoilage
and disease
2. Because a single transgenic plant cell can be
grown in culture to generate an adult plant,
plants are easier to engineer than most
animals
B. The Ti plasmid, from the soil bacterium
Agrobacterium tumefaciens, is often used
to introduce new genes into plant cells.
1. The Ti plasmid normally integrates a segment
of its DNA into its host plant and induces
tumors.
C.Foreign genes can be inserted into the
Ti plasmid using recombinant DNA
techniques
-recombinant plasmid put back into
Agrobacterium, which then infects plant
cells, or introduced directly into plant cells,
only used in dicots (two seed leaves)