Download Chapter 14: DNA Technologies

14
DNA Technologies
Lecture Outline
I.
Recombinant DNA methods grew out of research in microbial genetics
A. Research on bacteriophages laid the foundation for recombinant DNA
methods
B. Restriction enzymes are "molecular scissors"
1. Restriction enzymes cut DNA in areas of specific base pair sequences that
are palindromic
2. Staggered cuts in palindromic regions leave strands with complementary
("sticky") ends
3. Segments of DNA with "sticky" ends can be joined with DNA ligases
4. Restriction enzymes vary in the number of DNA bases they recognize
a) A restriction enzyme that recognizes a large number of bases has a low
probability of cutting in an inappropriate spot and is useful in research
on entire chromosomes
C. Recombinant DNA is formed when DNA is spliced into a vector
1. Bacteriophages or plasmids are common vectors
a) Foreign DNA and vector DNA are cut with the same restriction
enzyme, making them useful tools
b) Using antibiotics, the transformed colonies can be identified
2. The vector can be introduced into the host cell by transformation
a) Transformation involves making the bacterial cell wall permeable to
the plasmid
b) Plasmids often carry genes for resistance to antibiotics
c) Plasmids can carry a DNA segment of smaller than 10 kb
d) Bacteriophage vectors can carry segments of up to 23 kb
3. Another type of vector is a cosmid cloning vector, which is a combination
vector with characteristics of both bacteriophages and plasmids
4. Bacterial artificial chromosomes (BACs) accommodate larger fragments of
DNA (up to 200 kg of extra DNA)
a) These are particularly of importance in the processes used in the
Human Genome Project
5. Engineered viruses can also be used as vectors in mammalian cells
D. DNA can be cloned inside cells
1. A genomic library contains fragments of all of the DNA in the genome
a) A chromosome library contains all of the DNA fragments isolated
from the individual chromosomes of the organism of interest
b) It is easier to isolate a gene of interest from a chromosome library than
a genomic library
2.
Cloning techniques provide the means for replicating and isolating many
copies of a specific recombinant DNA molecule
a) After the restriction enzyme cuts the DNA to be cloned, the fragments,
all with complementary ends, are of different lengths
b) The restriction enzyme cuts both the plasmid and the DNA of the
organism with the gene of interest
c) The 2 types of DNA are mixed, which allows pairing of the
complementary ends
d) The gene is then cloned in E. coli
e) The cells are incubated on a medium with antibiotics
f) The selection scheme allows only the cells with the recombinant genes
to survive, as plasmids often confer resistance to antibiotics
g) Each recombinant bacterium gives rise to a visible colony that is clonal
3. A specific DNA sequence can be detected by a complementary genetic
probe
a) A probe is a radioactively labeled segment of RNA or single-stranded
DNA that is complementary to the target sequence
b) The probe hybridizes to the base pairs of the sequence of interest
c) The colony with the hybridized sequence is now radio-actively labeled
and can be detected by x-ray film
4. A cDNA library is complementary to mRNA and does not contain introns
a) Bacteria cannot remove introns, so reverse transcriptase is used to
make a DNA copy of the mRNA
b) The complementary DNA (cDNA) can then be inserted into the DNA
of a plasmid or virus vector
c) Comparison of cDNA and genomic DNA allows identification of
introns and exons
d) cDNA sequences are useful because they lack introns, and bacteria are
therefore able to read them and produce a functional protein product
E. The polymerase chain reaction (PCR) is a technique for amplifying DNA in
vitro
1. PCR allows amplification of a small amount of targeted DNA in a short
time
a) This was developed by Kary Mullis in 1985, for which he received the
Nobel Prize in 1993
2. A DNA sequence is first heated to separate the strands
a) The solution is then cooled
b) It is then exposed to DNA polymerase and specific primers to produce
2 identical strands
3. The process is repeated over and over to produce millions of copies of the
original DNA strand
4. A DNA polymerase (Taq) from a thermophilic bacterium (Thermus
aquaticus) is useful, as it is not adversely affected by the heating process
5.
The use of PRC is virtually limitless: from forensics to analyzing fossils
a) One minor drawback is the sensitivity of this process, and samples can
easily be contaminated
b) However, samples with even the tiniest amount of DNA can be
amplified and analyzed
F. Gel electrophoresis is the most widely used technique to separate
macromolecules
1. DNA and RNA are negatively charged and move through a gel at varying
speeds due to different molecular weights (lengths)
2. DNA fragments are often denatured, transferred to a membrane, and
incubated with a radioactive DNA probe
a) This resultant "blot" is used for autoradiography or chemical
luminescence and further studies
b) This type of blot is called a Southern blot after its inventor, E.M.
Southern
(1) The Southern blot can be used to diagnose genetic disorders
c) Blots used for RNA, separated by electrophoresis, are called Northern
blots; the Western blot is used for protein or polypeptide molecules
(One well known use is to detect antibodies, such as antibodies to HIV:
the test for AIDS)
G. A great deal of information can be inferred from a DNA nucleotide sequence
from the chain termination method of DNA sequencing
1. This method was developed by Sanger and Gilbert in 1974, for which they
received the Nobel Prize in 1980
2. Incorporation of dideoxynucleotides allows the investigator to determine
the sequence of bases
a) Dideoxynucleotides are modified synthetic nucleotides that prevent
elongation of the DNA strand
b) Reaction mixtures are prepared containing DNA polymerase,
radioactively labeled primers, 4 deoxynucleotides, and only 1 of each
of the 4 dideoxynucleotides (ddATP, ddCTP, ddGTP or ddTTP)
c) Fragments of varying length are therefore formed in each mixture,
which may be separated based on length
3. Much of this sequencing is now automated and can be done rapidly using
fluorescent dyes instead of radioactive labels
4. A machine can now decode 1.5 million bases in 24 hours
a) The Human Genome Project, began in 1980, resulted in the sequencing
of 3 billion base pairs of the human genome in 2001
b) By 2003, genomes of over 100 organisms were sequenced
c) The sequence of E. coli is now completely known, and the partial
sequences of many eukaryotes is being determined by many private
and public groups on an international scale
d)
5.
II.
Information is being kept on large computers in databases, many of
which are internet accessible
Restriction fragment length polymorphisms (RFLPs) are a measure of
genetic relationships
a) A restriction enzyme cuts DNA into fragments, which are of different
lengths in different individuals
b) A DNA fingerprint produced by gel electrophoresis of these fragments
shows a characteristic and individual pattern of banding
c) This technology is particularly important in determination of paternity
and in forensics
Genetic engineering has many applications
A. Genetic tests may now determine if a person has a genetic mutation
B. Gene therapy is the use of specific DNA to treat a disorder by actually
correcting a genetic problem
C. Human insulin produced by E. coli was one of the first engineered proteins
1. Human growth hormone and many other products are now produced by
E. coli
2. Other recombinant products are TPA, TGF-B, human blood clotting factor
VIII, and DNase
3. Recombinant technologies are increasingly being used to produce safer and
more effective vaccines
D. Additional engineering is required for a recombinant eukaryotic gene to be
expressed in bacteria
1. Introduced genes are not necessarily expressed by bacteria
2. Combining a strongly expressed bacterial promoter sequence with the gene
of interest may increase expression
3. Introduction of a cDNA copy may improve production
E. Transgenic organisms have incorporated foreign DNA into their cells
1. Viruses may be used as vectors to introduce DNA into animal or plant cells
2. Transgenic animals can produce genetically engineered proteins
a) Transgenic animals may be produced by injecting the DNA of interest
into a fertilized egg cell or an embryonic stem cell (ES cells)
b) Some transgenic animals produce a useful protein that can be
extracted from their milk (e.g., sheep)
c) Retroviruses may be used as vectors
3. Gene targeting, in which a researcher "knocks out" or inactivates a gene to
discover the role of the gene
a) Much research has been done on mice due to their genetic similarity
to humans
b) This technology is used to introduce genes to ES cells
c) There are about 3000 strains of "knock out" mice under current study
4.
5.
III.
Mutagenesis screening involves treating male mice with chemical
mutagens and breeding them, scanning for unusual phenotypes
a) This doesn’t knock out the gene, rather it causes a small change,
allowing the researcher to see what the result of a tiny change would
be
Transgenic plants are increasingly important in agriculture, and are being
developed to be resistant to insect pests, draught, heat, cold, herbicides,
salty, or acidic soil
a) Crops can be developed that are more nutritious and enriched in
vitamins A or B, or beta-carotene; although it is hard to increase
protein content
b) Agrobacterium tumefaciens (the crown gall bacterium) is often used in
plant biotechnology
(1) The Ti (tumor-inducing) plasmid is used as a vector
(2) The Ti vector primarily infects dicot plants
c) Genetic "shotguns" may be used for monocots
(1) Most of our important agricultural plants are monocots (corn, rice,
wheat)
d) Current research involves manipulation of the chloroplast DNA
(1) Questions remain whether it should be labeled, although the
majority of scientists believe that transgenic crops are safe
Safety guidelines have been developed for recombinant DNA technology
A. Concerns about the accidental release of genetically engineered microbes has
been groundless to date (see Lecture Enrichment)
B. Stringent restrictions exist in areas of biotechnology that are not well known,
or where potential for hazards are known
Research and Discussion Topics

Discuss current advances in testing for genetic conditions that have been made
possible using genetic technology. For example, a test that is reasonably reliable
allows people to find out whether they have the gene for Huntington disease (HD).
Would you be tested if a member of your family developed HD?

Research the various regulations in the United States on genetic experimentation.
Compare and contrast the regulations using prokaryotes, plants, and animals in
genetic manipulation.

Research various careers in biotechnology. What educational requirements are
necessary for these jobs? Where in the United States are most of these
biotechnological companies located?

What is the current status of the patent law associated with biotechnology? Can you
get a patent for a genetically engineered bacterium? A plant? An animal, like a
genetically altered mouse?

What is the current legal status of allowing DNA evidence in the courtroom?
Investigate current trials that have involved DNA evidence.

A recent survey showed that over 50% of Americans think altering human genes is
unethical. As a scientist, how might you respond to this attitude?