Download CH 20 DNA TECHNOLOGY - Ed W. Clark High School

AP Biology
Techniques and Applications of Biotechnology
Chapter 20
I. UNDERSTANDING AND MANIPULATING GENOMES
A. Recombinant DNA is DNA in which nucleotide sequences from two different sources are combined
into one DNA molecule.
B. The methods for making recombinant DNA is called genetic engjneering
C. Biotechnology allows for the manipulation of organisms and their components to make useful
products.
II. USES FOR GENE CLONING
A. make copies of a particular gene
B. make a protein product based on a particular gene
III. USING RESTRICTION ENZYMES AND RECOMBINATION
A. restriction enzyme – enzyme that cuts at specific sites on DNA/hundreds are used today/bacteria
protect own DNA by methylating (adding a CH3) its DNA so it’s not cut by the enzyme
B. restriction fragments - remaining DNA fragment after cut by enzyme
C. sticky ends – single strand DNA end part of the restriction fragment
D. DNA ligase - glues together DNA fragments by producing phosphodiester bonds
IV. RECOMBINATION USING RESTRICTION ENZYMES
1. restriction enzyme cuts at recognition site
2.sticky ends are produced
3. foreign DNA fragment produced by the same restriction enzyme is inserted
4. DNA ligase glues together fragment into dsDNA
V. RECOMBINANT DNA VECTORS –VOCABULARY
A. cloning vector-DNA that carries foreign DNA into a cell and replicates there
B. bacterial plasmid-circular DNA of a bacteria that is often used as a vector
VI. STEPS FOR CLONING A EUKARYOTIC GENE IN A BACTERIAL PLASMID
1.isolate the plasmid from a bacterium and the DNA from human cells that contain the gene
2. cut both with the same restriction enzyme
3. mix the cut plasmids and DNA fragments and add DNA ligase to seal them together
4. introduce the DNA into bacterial cells
5. allow the bacteria to grow
VII. IDENTIFYING CLONES CARRYING THE GENE OF INTEREST- NUCLEIC ACID
HYBRIDIZATION
1. Filter paper pressed against the master plate transfers cells to the bottom of the paper. Xs are used to
mark the location of each colony.
2. Filter is treated to denature DNA to allow the molecules to stick to the filter. Complementary
radioactive probe molecules are incubaterd with the paper
3. Radioactive areas are exposed to photographic film to show up where the hybridized DNA appears
4 . The reference points on the film and on the master plate are aligned so the colonies with the gene of
interest are located.
5. Now a genomic library is created making copies of the colonies containing the gene of interest.
VIII. Genomic Libraries- a complete set of plasmid clones, each carrying copies of a particular segment
of DNA Sets are maintained and obtained from research facilities around the world.
IX. AMPLIFYING THE GENE OF INTEREST- POLYMERASE CHAIN REACTION (PCR)
A. A popular technique used for quicker and more selective sequencing. It can make billions of copies in
a few hours in a test tube.
1. obtain starting DNA with desired sequence
2. heat strands to separate
3. cool strands to allow primers to anneal
4. add DNA polymerase from a high heat-resistant bacterium (Taq)
5. two new strands result
6. repeating the above gives exponential growth in the number of new DNA formed
7. note there are errors that occurs with the more Replication, so there is a limit
X. DNA ANALYSIS AND GENOMICS
A. Restriction fragment analysis-detection of differences in the nucleotide sequences of DNA molecules
B. GEL ELECTROPHORESIS (pg 393) This technique uses a gel to separate nucleic acids or proteins
by size or electrical charge (negative charge is carried on phosphate end which will travel towards a
positive electrode)
1. take several mixtures of various fragments of DNA (note that DNA is negatively charged so it moves
toward to anode end)
2. add an electrical current to create the positive and negative end, longer fragments travel slower than
shorter ones and will see this as a banding pattern
3. stain gel then put under UV light
4. if banding patterns match, the same segment of DNA was placed into each well
C. SOUTHERN BLOTTING
1. run a gel electrophoresis on samples to produce a smear of bands
2. place the gel in an alkaline solution with nitrocellulose paper on top and paper towels on top of that
3. as the alkaline solution rise up through the layers, the DNA is transferred and denatures to form
ssDNA on the nitrocellulose paper in the same pattern
4. place paper and DNA probes (fluorescent labeled ssDNA which will hybridize DNA sequence of
choice) in a bag to anneal
5. rinse away unattached probes and compare sequences – same DNA will have same banding pattern
D. GENETIC MARKERS (noncoding DNA)- differences in the fragment banding patterns are called
restriction fragment length polymorphisms (RFLPs) and are as genetic markers. These RFLPs are
inherited and are used to make linkage maps. Ultimately it was these markers that was helpful in
completing the Human Genome Project
XI. MAPPING AN ENTIRE GENOME
A. genetic mapping is used – create a linkage map with markers spaced through the chromosome
The markers are now all in order
B. Physical mapping is now used to measure physical distance. The DNA is cut up into fragments. The
fragments are then put into order as they would appear on the chromosome. Common vectors would be
yeast or bacterial sources which are smaller and easier to handle. Often bacteriophages and plasmids are
used for the same purpose. The smaller fragments are easier to put in order.
C. DNA sequencing can be determined by the small fragments to assemble partial sequences of the bases
themselves. Often the “shotgun approach” is used
1. cut the DNA into overlapping fragments
2. clone the fragments using phages or plasmid vectors
3. sequence each fragment
4. order the sequences using a computer(s)
XII. GENOMICS- the study of whole sets of genes and their interactions
A. Identification of protein-coding genes by a computer data base
B. Gene function can be determined by disabling the gene and seeing what happens to the organism.
C. Groups of genes can be studied together to determine how they maintain a whole organism. DNA
microarray assay
XIII. APPLICATIONS OF DNA TECHNOLOGY
1. Medical applications for diagnosis of diseases by analyzing the RFLPs (restriction fragment length
polymorphisms using Southern Blotting)
2. Gene Therapy can be used to alter an individual’s genes to help treat diseases by inserting a normal
allele of a defective gene . Retroviruses have been used as vectors for this research in bone marrow
diseases. Bone marrow cells are ideal because they reproduce throughout a person’s life.
3. Pharmaceutical products such as insulin and growth hormone
4. Forensic evidence
5. Environmental cleanup using bacterial to help remove heavy metals or oil in clean ups. Bacteria are
also commonly used in sewage treatment plants to break down components.
6. “Pharm” animals are transgenic (having genes from different species) to produce a better quality
animal. (safe for consumption if someone has an allergy?)
7. Genetic engineering in plants- common vector is the plasmid called Ti (not treasure island) Can
make plants pest resistant or improving nutritional value. One example is golden rice which has more
Vitamin A than regular rice and for half the world’s population that relies on rice as their food staple, it
could lead to healthier children.
XIV. ETHICAL QUESTIONS -Public concern over these “genetically modified organisms” is that the
safety for human consumption is questioned. BTW Most salmon you eat now is a GM version. Plants give
us the most concern. If these GM organisms get out into the wild and exchange genes, they could transmit
their resistance to pesticides to create “superweeds” Agencies in the world are closely monitoring these
developments.
Vocabulary Terms
Biotechnology
Cloning vector
DNA fingerprint
DNA microarray assays
Gene cloning
Genetic Engineering
Genomic library
Human genome project
Nucleic acid hybridization
Physical map
Recombinant DNA
Restriction fragments
Restriction fragment length polymorphisms (RFLPs)
Southern blotting
Clone
Denaturation
DNA ligase
Gel electrophoresis
Genetically modified organisms GMOs
Gene therapy
Genomics
Linkage map
Nucleic acid probe
Polymerase chain reaction PCR
Restriction enzymes
Restriction site
Sticky end
transgenic