Download Micro Chpt. 10 Notes

Micro CHAPTER 10 Notes
1. Genetic engineering is the manipulation of the genetic material of a cell to alter function. Examples of
genetic engineering include creating knockout mice to study genes, creating transgenic microbes to
produce medicinal gene products (e.g. human insulin), and creating vaccines or gene therapy
vectors. Biotechnology is the use of live organisms, including genetically modified organisms, and
their biochemical products to benefit mankind. Biotechnology includes the production of medicines
and enzymes from microbes (both natural and recombinant), the alteration of crops and animals to
encourage growth and disease resistance, and the study of human genes and relationships to
disease. The genome is a total compliment of DNA and the instructions, or genes, carried within. A
chromosome is an individual unit of DNA; the bacterial genome typically consists of one and the
human genome contains 23 pairs. A gene is an individual cellular instruction within a DNA sequence
and typically encodes a protein. DNA, or deoxyribonucleic acid, is the molecule that carries genetic
information in all cells and some viruses. RNA, or ribonucleic acid, is transcribed from DNA and
typically carries the code of one gene or a few genes to be transcribed. It may also play a role in
protein translation (tRNA and rRNA).
2.
a. Complimentary strands of DNA are held together by base pairing, or hydrogen bonds that join
complimentary bases on opposite strands. Denaturation with use of enzymes or high
temperatures breaks the hydrogen bonds to separate the strands of DNA, and renaturation, with
use of enzymes or lower temperatures, reforms the bonds to recreate the double helix.
b. Denaturation is an important part of replication and transcription and allows single-stranded DNA
to serve as the template for both processes. Denaturation allows hybridization or annealing with
primers, probes, and individual nucleotides both in vivo and in vitro.
c.
A Southern blot test involves the hybridization of a labeled DNA probe to complimentary DNA
sequences on a solid support surface. Hybridization requires a single-stranded DNA target (i.e.
denatured double-stranded DNA).
d. PCR can be used to amplify numerous sequences within a genome. After the separation of PCR
products by gel electrophoresis and the subsequent transfer to a support membrane, the DNA
can be probed to identify the desired DNA sequences that were amplified.
3.
a. A restriction endonuclease is a bacterial enzyme that breaks phosphodiester bonds in doublestranded DNA at palindromic sequences. They are used as a mechanism for bacteria to digest or
restrict foreign DNA entering the cell.
b. A palindrome is a double-stranded DNA sequence that reads the same on the top strand (5’3”)
as it does on the complimentary strand in the antiparallel direction.
EcoRI GAATTC
CTTAAG
c.
HindIII AAGCTT
TTCGAA
SmaI
CCCGGG
GGGCCC
One can cut a circular piece of DNA to linearize it. Or one can cut a linear piece of DNA at two
restriction sites that produce a compatible or “sticky” end and then join the end with DNA ligase to
produce a circular DNA molecule (see Figure 10.1).
d. Because of sequence differences in genomes, the location of sequence-specific restriction sites
(palindromes) may vary between genomes. When digested with a particular restriction enzyme,
the DNA will be cut into a pattern of different size fragments that can be resolved and
differentiated by gel electrophoresis and Southern blot analysis. These are called restriction
fragment length polymorphisms (RFLP), meaning “many forms.”
Chapter 10
4. Electrophoresis involves placing a sample of DNA fragments into a solid gel matrix, typically agarose
or polyacrylamide, immersed in an aqueous buffer that conducts electrical current. When an electrical
current is passed through the gel, positive and negative poles are set up on either end of the gel.
DNA, because it is highly negatively charged, will migrate toward the positive pole of the gel. The
matrix will retard the movement of larger DNA fragments so the DNA fragments of different sizes will
be separated, where the smallest fragments migrate the furthest. The DNA fragment in lane 1 of the
gel in Figure 10.2b is approximately 2000 base pairs. Oligonucelotides are short single-stranded
DNA sequences that are chemically constructed in vitro or can be produced by DNA digestion and
purification. Oligos can be used as primers for PCR, as probes for Southern hybridization, or for sitedirected mutagenesis of DNA.
5.
a. With use of an mRNA template, a poly(dT) primer (underlined sequence in 5c), DNA nucleotides,
a retroviral reverse transcriptase enzyme, and the proper buffer and incubation conditions, a DNA
copy is created. This process is also known as reverse transcription.
b. The DNA produced by reverse transcription of mRNA is called complimentary DNA (cDNA).
c.
Since mRNA in eukaryotes contain a 3’ polyA tail but tRNA, rRNA, and other RNA do not, one
can use a poly(dT) primer to selectively create a cDNA library that only contains the spliced (no
introns), protein-encoding regions of the genome.
6.
a. DNA synthesizers chemically join nucleotides to produce single-stranded DNA molecules, or
oligonucleotides, that have a defined sequence. DNA sequencers determine the order of bases,
or sequence, in a given stretch of DNA.
b. Figure 10.5 illustrates the steps in Sanger DNA sequencing. Briefly, a primer is used to start
DNA replication at a specific site on the template DNA. DNA Polymerase inserts complimentary
deoxyribonucleotides (dNTP) to create a new DNA strand. In each of four separate reaction
tubes, a different dideoxyribonucleotide (ddNTP) is added to the reaction mixture. When a
ddNTP is incorporated into a DNA chain, it stops replication and a DNA fragment of a defined
size is produced. For example, if ddATP is present, it will terminate replication of the template at
any position where there is a thymine (T), the complimentary base. When the DNA fragments
from this reaction are separated by gel electrophoresis, the position of all T will be revealed.
Likewise, reaction tubes containing ddCTP, ddGTP, and ddTTP will reveal the positions of G, C,
and A, respectively, by displaying length polymorphisms that differ by one nucleotide each.
7.
a. To perform PCR to amplify a sequence of DNA, high heat (95ºC) is used to denature the doublestranded DNA template. The temperature is then reduced (50-60ºC) to allow annealing of
primers to the single-stranded templates, and then the temperature is raised slightly (72ºC) to the
optimal polymerization temperature for Taq DNA Polymerase. Following synthesis, the process
starts again with heat denaturation, and multiple cycles yield many copies of the template and of
each DNA copy.
b. The primer serves to provide sequence- or target-specific amplification and to define the
boundaries of the amplified sequence. Taq Polymerase is a DNA replication enzyme that
withstands the high temperatures utilized during cycling.
c.
PCR is unlikely to amplify contaminating bacterial DNA in a human DNA sample because the
primers will be designed to be specific for the human sequence(s) of interest and will not anneal
under the proper conditions to other nonspecific sequences.
d. With knowledge of the sequences flanking a gene of interest, primers can be designed to anneal
and amplify that region specifically, even while contained in an entire genome’s worth of DNA.
Optimization of primer concentration and annealing temperature should be considered.
8.
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a. Plasmids and bacteriophages make good cloning vectors because, in addition to being small
enough to have their entire sequence known, they have the ability to encode their own replication,
accept donor DNA up to a certain size (multiple genes), transfer DNA into live cells (e.g.
transformation or transduction), and be easily manipulated in the lab.
b. Plasmids are smaller DNA molecules that can carry DNA inserts up to 10 kb in size. Cosmids
can hold much larger (45 kb) inserts. Bacterial artificial chromosomes (BACs) can carry as much
as 300 kb of insert DNA. All are useful for cloning and expression in bacteria. Yeast artificial
chromosomes are useful for genetic manipulations of yeasts. Bacteriophage, Agrobacterium,
bacculovirus, and adendovirus are useful for introduction of DNA into bacterial, plant, insect, and
human cells, respectively.
Vectors may contain selectable markers (e.g. antibiotic resistance genes) so that cells that take
up the DNA can be easily identified. Some vectors also contain genes that create fusions (e.g.
beta-galactosidase or green-fluorescent protein) to track gene expression, or genes that direct
chromosomal insertion (e.g. transposons).
9.
a. Recombinant DNA technology involves the insertion of foreign DNA into vector DNA for the
purposes of cloning or expression in a host organism.
b. Bacteria are good cloning hosts not only because they grow rapidly, but because DNA is easily
introduced by a number of methods and species such as E. coli are well defined in terms of
genetics and metabolism.
c.
To clone a gene, the sequence of interest can be excised from a genome with use of restriction
enzymes or simply amplified with use of PCR. The vector is linearized with a restriction enzyme
and the insert with compatible ends is ligated into the vector. The vector is introduced (e.g.
transformed) into a bacterial cell. When the transgenic bacterium is cultured under selective
conditions, not only is the recombinant DNA copied as cells reproduce, but each individual cell
may carry as many as a hundred copies of the DNA.
d. Once cloned, the gene can be sequenced, purified and used as a probe, mutated or altered by
numerous techniques, and expressed individually or as a fusion protein. The gene product can
then be purified.
e. Recombinant DNA technology can be used to study gene expression or function (e.g. knockout
mice), express large quantities of a foreign protein (e.g. human insulin production in bacteria),
introduce new functions into a species (e.g. pest resistance in crops), increase protein production
in a species (e.g. growth hormones in fish), and repair defective genes (e.g. gene therapy in
humans).
10. Vectors that encode drug resistance in bacteria can be selected for by incubating the culture in the
presence of the drug. Only those bacteria that have acquired the recombinant plasmid will be
resistant and grow.
11.
a. A DNA fingerprint or profile is a pattern of DNA fragments separated by gel electrophoresis and
represents the restriction fragment length polymorphisms that differentiate individuals.
b. DNA fingerprinting is being used in medicine to identify sources of infectious outbreaks, in law to
convict or exonerate individuals of crimes, in the military to identify remains of soldiers killed or
missing in action, and in human biology to identify carriers of disease.
12. One scenario might involve identifying an unknown microbe from a sample. If there were only a few
viruses in the sample, using the Southern blot technique alone would probably not yield enough
genetic material to show up on a blot. By first subjecting the specimen to PCR, the genetic material
could be greatly amplified, so there would be enough DNA for the Southern blot hybridization to give
readable results. If the virus were an RNA virus, the isolated genetic material could first be converted
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to DNA with reverse transcriptase and then taken through PCR amplification. Again, this would yield
an identifiable type and amount of genetic material specific to the virus.
It would also be possible to start with a Southern blot of a known sample and then cut one of the
desired bands out of the gel, then use PCR to amplify the band so that a large amount of that specific
segment of DNA is obtained. This might be one of the steps in sequencing one part of a genome or
in making multiple copies of a DNA sequence without the need for recombinant DNA technology.
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