Chapter 9- Biotechnology and Recombinant DNA (EXAM 2) Flashcards
1
Q
The manipulation of living
organisms, or cell components to produce useful products.
A
Biotechnology
2
Q
Products made by biotechnology
A
Foods, antibiotics, vitamins, enzymes
– Pest resistant crops
– Bacterial strains for waste treatment, environmental oil
clean-up
– Limited to a cell’s own products until the 1980’s
3
Q
procedures that are used to join together (recombine) DNA segments in vitro
A
Recombinant DNA (rDNA) technology/genetic engineering
4
Q
1) Population of cells arising from a single parent cell
2) Processes used to create copies of DNA fragments
A
clone
5
Q
The production of exact copies (_______) of a particular gene or DNA sequence using genetic
engineering techniques.
A
gene cloning; cloning
6
Q
Describe the process of gene cloning
A
almost always with E.coli
- Vector, such as a plasmid is isolated
- DNA is cleaved by an enzyme into fragments
- Gene is inserted into plasmid
- Plasmid is taken up by a cell such as a bacterium
- cells with gene of interest are cloned depending on the goal
7
Q
What is the goal of gene cloning?
A
- either to make copies of the gene
2. or to make protein product of the gene
8
Q
When copies of the gene are harvested what can they be used for?
A
The gene itself is of interest.
- Plasmid borne genes are
easily manipulated
- Gene for pest resistance is inserted into plants
- Gene alters bacteria for cleaning up toxic waste
9
Q
When the copies of the gene make a protein product, and the desired proteins are harvested; what can they be used for?
A
- The product of the gene is
of interest. - Cloning human growth hormone was an early success
- amylase, cellulase, and other enzymes prepare fabrics for clothing manufacture
- human growth hormone treats stunted growth
10
Q
in nature organisms with
characteristics that enhance survival are more likely to
survive.
A
natural selection
11
Q
Why are bacteria good subjects to study natural selection?
A
Bacteria are a common research subject when studying evolution and adaptation because some colonies of bacteria can produce several generations in one day, letting researchers see a “fast forward” version of evolution and natural selection.
12
Q
humans select desirable breeds of animals or strains of plants
A
artificial selection
13
Q
provide examples of artificial selection
A
a farmer chooses high milk producing cows for breeding
- Pure bacterial cultures with favorable characteristics
can be selected
- beer brewing (efficiency, taste, alcohol content)
- antibiotic producing bacterial strains (also elevated
expression)
14
Q
a tool for biotechnology
A
mutagens
15
Q
_______________ can be used to increase the chances of obtaining a
desired strain
- Radiating _________ generated a strain that
produced 1000x penicillin
A
random mutagenesis (mutagen exposure); fungus
16
Q
a mutation created at a defined site in a DNA molecule
A
site directed mutagenesis
17
Q
Why is site directed mutagenesis useful?
A
Rather than screening/selecting for mutants, site directed mutagenesis (a mutation
created at a defined site in a DNA molecule) can be used to make a specific change in a
gene
18
Q
Cut DNA at defined positions close to or within their recognition sequences
A
Restriction enzymes
19
Q
What is the cutting frequency of restriction enzymes?
A
typically recognize 4-, 6-, or 8-base sequences
20
Q
Do restriction enzymes cut the same way each time?
A
Yes
21
Q
Some produce _______ ends, others produce ___________ (sticky) ends
A
blunt; staggered
22
Q
can be used to join two pieces of DNA with complementary ends
A
staggered ends
23
Q
Bacterial source of BamHI
A
Basicillus amyloliquefaciens
24
Q
Recognition sequence of BamHI
A
G|GATCC
GCTAG|G
25
Bacterial source of EcoRI
Escherichia coli
26
Recognition sequence of EcoRI
G|AATTC
| CTTAA|G
27
Bacterial source of HaeIII
Haemophilus aegyptius
28
Recognition sequence of HaeIII
GG|CC
| CC|GG
29
Bacterial source of HindIII
Haemophilus influenzae
30
Recognition sequence of HindIII
A|AGCTT
| TTCGA|A
31
Recognition sequence of HindIII
A|AGCTT
| TTCGA|A
32
Describe the process of Restriction Enzymes and Recombinant DNA
1. restriction enzyme cuts dsDNA at its particular recognition sites
2. These cutes produce DNA fragment with two sticky ends
3. when two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing
4. the joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur
5. The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of recombinant DNA
33
Compatable cohesive ends
Echo slide 13 (unclear)
34
Why do bacteria produce restriction enzymes?
They RESTRICT the ability of foreign DNA (such as
bacteriophage DNA) to infect/invade the host
bacterial cell by cleaving it)
35
In bacteria, how is host DNA modified to protect them against bacteriophages?
The host DNA is MODIFIED by methylation of their
| sequences at C or A nucleotides
36
```
This modification
protects the bacterial
host DNA from
degradation by its own
restriction enzyme
• Called _________ ___________ system
```
restriction modification
37
Autonomously-replicating DNA used to carry the desired gene to a new cell
vectors
38
Two types of molecules that can be used as vectors. What determines which type will be used in the study?
Plasmids and viruses can be used. (choice depends on organism receiving the gene and size of cloned DNA)
39
primary vectors in use. Easy to manipulate
plasmids
40
accept larger pieces of foreign DNA
viruses
41
types of viruses used to insert correctives genes into human cells
retroviruses, adenoviruses, and herpes viruses
42
Are there natural mammalian origins (ORIs)?
no (echo-need clarification slide 15)
43
What are some necessary properties for vectors?
- need to self replicatate
- must be a small size that facilitates manipulation outside the cell
- must be able to avoid destruction by host nucleases (ie. must be circular)
- must be able to carry a selectable marker gene (ie. antibiotic resistance or auxotrophic marker)
44
Would marker genes that can be phenotypically screened be useful?
Slide 16- Echo
45
An E.coli plasmid vector used for cloning
Echo slide 17
46
can replicate in at least two different species
shuttle vectors
47
Shuttle vectors require ________ _______ _________ and _______ __ __________. Provide some examples.
```
requires suitable
selectable markers and
origins of replication
(E. coli/yeast, E. coli/
*mammalian*, E. coli/
fungi, E. coli/plant, E.
coli/other bacteria)
```
slide- 18 clarify
48
distinguishing vector self-ligation from insert ligation
slide 19-clarify
49
A molecular technique that allows for the detection of
| successful ligations in vector based cloning
the blue white screen
50
Describe the blue white screen
1. plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasmid has the genes for lactose hydrolysis (the lacZ gene encodes the enzyme B-galactosidase) and ampicillin resistance.
2. Foreign DNA will insert into the lacZ gene. The bacterium receiving the plasmid vector will not produce the enzyme B-galactosidease if foreign DNA has been inserted into the plasmid
3. The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant
4. All treated bacteria are spead on a nutrient agar plate containing ampicillin and a B-galactosidease substrate and incubated. The B galactosidase substrate is called X-gal
5. Only bacteria that picked up the plasmid will grow in the presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound. The indgo turns the colines blue. Bacteria that cannot hydrolyze X gal produce white colonies.
51
Enzymatic method to amplify (make multiple
| copies) a piece of DNA to detectable levels
polymerase chain reaction
52
what is PCR useful for?
```
This is useful for
– Cloning a piece of DNA
– Sequencing DNA
– Diagnosing genetic diseases (i.e. restriction analysis)
– Detecting pathogens
```
53
What is the process of PCR?
1) Incubate target DNA, primers, deoxynucleotides
and DNA polymerase at 94C for 1 minute.
This allows separation of the DNA strands
2) Primers attach to a single-stranded DNA during incubation at 60C for 1 min
3) incubate at 72C for 1 minute; during this time, two copies of target DNA are formed
4) repeat the cycle of heating and cooling to make two more copies of target DNA
54
define denaturation
a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent, temperature etc.
55
What is unique about the DNA polymerase used in PCR?
use a Taq polymerase that can withstand high temperatures (Echo slide 24 to clarify)
56
What is the significance of the temperatures and times chosen for each step?
Echo slide 25 to clarify
57
Thermocycler
echo slide 26
58
Robocycler
echo slide 27
59
How is the amplified product detected?
echo slide 28
60
How does rtPCR/ qPCR differ from standard PCR?
echo slide 28
61
what is rt-PCR?
echo slide 28
62
What are the three phases of PCR? Describe these three phases.
1. exponential
2. Linear
3. plateau
Echo slide 29
63
What is the taqman method?
TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.[2] As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. The probe and primers actually bind to SS DNA. The levels of flourescence are measured after each cycle.
64
What is an alternative method to the Taqman method? Describe this method
SYBR green (echo slide 30)
65
Describe the process of RT PCR
reverse transcriptase is used for the synthesis of the first cDNA strand. Elongation. Denaturation: inactivates RT and separates the strands. Then, sequence specific primers and Taq polymerase are used for PCR amplification of a gene-specific fragment.
66
What primers are used in RT PCR?
echo slide 31
67
Most bacterial mRNAs have short or no ____________
polyA tails
68
What are some methods of transformation?
* Electroporation
* Chemical Transformation
* Protoplast fusion
* Gene gun
* Microinjection
69
define transformation
ways to insert foreign DNA into cells
70
What is the difference between transformation and transfection?
Both processes describe the addition of genetic material into cells using various techniques. Transformation is here mostly used for bacterial work (transforming plasmids for example), while transfection is almost exclusively used for eukaryotic cells.
71
What method of transformation is this: A controlled short (millisecond) but powerful electrical pulse induces temporary hydrophilic pores in the cell membrane; DNA can then enter the cell
• Generally applicable to all
cells
electroporation
72
In electroporation, what needs to be done to organisms with cell walls?
some organisms with cell walls may require prior conversion to protoplasts (plant cells and algae?)
(Not necessary for bacteria or yeast)
73
Which method of transformation is this:
• For E. coli, cells are incubated in ice-cold calcium chloride, DNA is added, then given a mild heat shock
• Similar method for yeast except lithium chloride is used
• heat shock (42C) thought to work by transiently opening gated membrane channels
chemical transformation
74
In chemical transformation, what is the process used for bacteria?
cells are incubated in ice-cold calcium chloride, DNA is added, then given a mild heat shock
75
In chemical transformation, what is the process used for yeast?
Similar method to bacteria except lithium chloride is used
76
Describe protoplast fusion method of transformation
• Takes advantage of the
fusion properties of protoplasts: fusion rate increases in the
presence of PEG
• most valuable in plant and algal cells
1. Bacterial cell walls are enzymatically digested, producing protoplasts
2. In solution, protoplasts are treated with polyethylene glycol
3. protoplasts fuse
4. segments of the two chromosomes recombine
5. recombinant cell grows new cell wall
77
takes advantage of the fusion properties of protoplasts
protoplast fusion
78
fusion rate of protoplasts increases in the presence of ______
PEG
79
What cell types are protoplast fusion most valuable in?
plant and algal cells
80
Microscopic gold or tungsten particles (approx. 1 µm) coated with DNA are
propelled into the cells
gene gun/ biolistic transformation
81
During biolistic transformation, what happens if a gold particle lands in the nuclues?
the genes elute off and may be incorporated in the chromosome
82
What types of cells or organelles are gene gun/ biolistic transformation used for?
plant cells, C. elegan cells, and yeast mitochondria. Other applications include bacteria, insect, animal and human cells
83
Transformation method that can be applied to the widest range of cell and tissue types.
biolistic particle bombardment
84
uses a glass micropipette to inject DNA into the cell
microinjection
85
What is microinjection not useful for?
impractical for bacterial and fungal cells.
86
a collection of DNA fragments of one organism, each carried by a plasmid or
virus and cloned in an appropriate host.
DNA libraries
87
What are the two types of DNA libraries and what do they contain?
Genomic library: contains DNA fragments
representing the entire genome of an
organism
cDNA library: contains only complementary
DNA molecules synthesized from mRNA
molecules in a cell
88
What is the purpose of making a genomic library?
The goal is to make a collection of clones
| large enough to ensure that at least one clone exists for every gene in the organism
89
What library size (# genome equivalents)
is necessary to ensure that all
sequences of the genome are
represented?
echo slide 39
90
What is the choice of restriction enzyme in making a genomic library?
echo slide 39
91
Genome to be stored
in library is cut up with
________ _______
restriction enzyme
92
is made from
mRNA by reverse
transcriptase
complementary DNA (cDNA)
93
cDNAs are then cloned into a _______ or ______ ______
plasmid or viral vector
94
```
Common for eukaryotic
libraries but ______ ______
may not be completely
reverse transcribed into
DNA
```
long mRNAs
95
What primer sequence will
allow reverse transcription
of all nuclear eukaryotic
mRNAs?
echo slide 41
96
Describe the process of making a cDNA library
1. a gene composed of exons and introns is transcribed to RNA by RNA polymerase
2. Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mRNA
3. Isolate mRNA from the cell and add reverse transcriptase
4. first strand of DNA is synthesized
5. the mRNA is digested by reverse transcriptase
6. add DNA polymerase to synthesize second strand of DNA
97
DNA can be made _________ on a solid matrix using a
| DNA synthesis machine
chemically (in a lab)
98
Two complementary ssDNA molecules can be
| synthesized and then hybridized to generate a dsDNA molecule
synthetic DNA
99
What are some limitations of synthetic DNA?
- less than 200 bases in length
- gene sequence will
be ambiguous when only the
protein sequence is available
although fragments can be ligated together, they are not
typically used for gene cloning
- more commonly used for primers and probes
100
How can you identify a clone from a library containing your gene of interest?
colony hybridization
101
Describe the process of colony hybridization
1. Obtain a master plate with colonies of bacteria containing cloned segments of foreign genes
1. make a replica of master plate on nitrocellulose filter
2. treat filter with detergent (SDS) to lyse bacteria
3. treat filter with sodium hydroxide (NaOH) to separate DNA into single strands
4. add radioactively labeled probes
5. probe will hybridize with desired gene from bacterial cells
6. Wash filter to remove unbound probe and expose filter to X-ray film
7. compare developed film with replica of master plate to identify colonies containing gene of interest
102
Can you name an alternative method that is available to identify a specific clone in a library?
echo slide 43
103
How can PCR be used to obtain a genomic or cDNA clone?
echo slide 44
104
Which cell types are used to make gene products?
bacteria (E.coli)
| yeast (S. cerevisiae, plant cells and whole plants, and mammalian cells
105
What are the advantages and disadvantages of using E.coli to make genes?
```
Advantages:
It is easily grown, can
carry plasmids and its
genomics are known
(i.e. inducible promoters)
```
```
Disadvantages:
- Need to eliminate
endotoxin from
products
- Does not normally
secrete products
(must lyse cells to
to get product)
```
106
What alternative bacteria is more likely to secrete?
echo slide 45
107
Easily grown, can carry
plasmids and its
genomics are well
known
Greater chance of
expressing eukaryotic
genes
More likely to correctly
modify eukaryotic
proteins than bacteria
Likely to continuously
secrete products
Saccharomyces
| cerevisiae
108
May express eukaryotic genes
easily
Low risk of product
contamination by mammalian
pathogens
Large scale low-cost production
plant cells and whole plants
109
May express eukaryotic genes easily
Well suited to make proteins for medical use
- secreted and low risk of toxins or
allergens
Harder to grow
mammalian cells
110
What are some therapeutic applications of rDNA?
human enzymes
subunit vaccines
DNA vaccines
gene therapy
111
examples of human enzymes made by rDNA
human insulin
- human hormone somatostatin (5 mg = 50,000
sheep brains or 8 L bacterial culture)
112
specific protein from a pathogen (purified from yeast or expressed as a viral surface protein)
subunit vaccines
113
injection of plasmids carrying genes for a specific
| pathogens antigens. The expressed protein then produces an immunological response
DNA vaccines
114
replace defective or missing genes
gene therapy
115
an early commercial success of rDNA technology (1978)
human insulin production
116
Human insulin subunits are ____ and ____ amino acids
21 and 30
117
Each subunit in the human insulin was made ________
synthetically
118
Describe the process of making a DNA vaccine?
1. gene for an immunogen
2. insert gene into an expression plasmid
3. transform bacterial cells, grow bacteria, and purify the plasmid DNA
4. Immunize with immunogen-expressing plasmid
119
What is the first approach to gene therapy?
replace or augment a faulty gene with a normal one (First used in 1990)
120
The first approach to gene therapy relies on finding a delivery system to carry the correct gene to the
affected cells. The gene must be delivered inside the ______ ______ and work properly for the _____ term without causing ______ _______.
target cells; long; adverse effects
121
most gene deliveries are performed by what?
```
most gene deliveries
are performed by adeno- and
retroviruses, plasmid vectors
and other strategies are being
tried
```
122
which of the gene therapy vectors is most promising?
adeno associated virus
| vectors are showing promise
123
What is the second approach to gene therapy?
A second approach is to inhibit expression of the defective gene by RNA interference
124
a eukaryotic biological process in which RNA molecules inhibit gene expression
RNA interference (RNAi)
125
what are the two small RNA molecules that are central to RNAi?
microRNA (miRNA) and small/ short interfereing RNA (siRNA)
126
RNAi is also called what?
gene silencing
127
How are the pathways similar and different? How is RNAi useful experimentally?
slide 52
128
How do you sequence an organism's genome?
one technique is to random shotgun sequence it
construct a gene library
closure phase
129
How does the process of random shotgun sequencing work?
1. isolate dna
2. fragment dna with restriction enzymes
3. clone dna in a bacterial artificial chromosome (BAC)
4. sequence dna fragments
5. assemble sequences
6. edit sequences; fill in gaps
130
How are the gaps determined?
slide 54
131
_______________: • Initiated in 1990 with the goal of sequencing the 3 billion
base-pair human genome in 15 years.
• NIH and DOE instituted the joint project in which 18
countries contributed.
• There was great skepticism that this could be accomplished
in a reasonable amount of time.
- helped by random shotgun sequencing and sequencing
advances
• The human genome draft was completed in 2003.
• Results:
- less than 2% encodes product, remainder is ______ DNA
(miRNA genes, short tandem repeats, introns, telomeres,
transposons, viral remnants, pseudogenes etc.)
• Should benefit research as well as diagnostics and
treatments of genetic diseases
human genome project; junk
132
the science of understanding the function of
| genes through computer assisted analysis
bioinformatics
133
DNA sequences are stored in a web based database known
as ________ (provided by the National Center for
Biotechnology Information (NCBI).
• Genomic information can be searched with computer
programs to find:
- specific sequences (i.e. _______)
- similar sequences in other organisms
- _______
GenBank; motifs; ORFs
134
What is proteomics?
slide 57
135
-starts with a phenotype and identifies the responsible gene
Forward genetics (classical genetics):
136
-starts with a particular gene and assays the effect of its disruption
Reverse genetics:
137
• A relatively new specialty of microbiology
• The study of microbes to to determine the path of
an outbreak, the identity of a criminal or the origin of
a particular strain of contagion or biological weapon.
- microbes as terrorist weapons (i.e. anthrax
enclosed in envelopes to sicken or kill victims)
- Microbes as a factor in cases of medical
negligence
- The deliberate infection of people with a
communicable disease
- Intentional food contamination.
• Must be done in a precise, methodical manner that
allows courts to draw conclusions from the data
Forensic Microbiology
138
```
Involved with the design
and manufacture of
electronic circuits and
devices built at the
molecular level (the
object has one
dimension that is in the
order of nanometers)
• Bacteria can make
molecule-sized particles
that could provide the
needed small wires and
components
```
nanotechnology
139
What is the benefit of using bacteria to produce nanospheres?
drug
targeting and
delivery
140
```
• The bacterium _________________
infects specific plants at
wound sites
• This bacteria contains a
naturally occurring plasmid
(Ti)
• A part of the Ti plasmid, called
T DNA, integrates into the
genome of the infected plant.
• The T DNA contains genes for
_______ synthesis and tumor
production (_______). ________
is a unique amino acid used by
the bacteria as a carbon and
nitrogen sources
```
Agrobacterium tumefaciens; opine; crown gall disease; opine
141
Ti plasmid also carries
| genes of opine ________
catabolism
142
In a plant cell, only a _________ strand enters the plant cell
ssT-DNA
143
describe a normal infection by agrobacterium
slide 64
144
What can the Ti plasmid be used for in plants?
Using the Ti plasmid as a vector for
| genetic modification in plants
145
What can agrobacterium not be used for?
Agrobacterium does not naturally infect grasses, so can’t be used to improve grains
such as wheat, rice, or corn
146
Describe the process of using the Ti plasmid as a vector in GMO plants
slide 65
147
• plants resistant to the herbicide
glyphosphate (“________’)
- herbicide inhibits an enzyme responsible
for synthesizing ________ amino acids
- mutant gene resistant to herbicide
selected in _________ bacteria then
introduced into crop plants
• _____ toxin gene has been introduced into a
number of plants (i.e. cotton and potatoes)
- insects that eat plants are killed
roundup; aromatic; Salmonella; Bt
148
What are some other agricultural traits that can be dealt with by genetic engineering?
resistance to other herbicides, male sterility, flower color,
modified fatty acids, virus resistance
149
What are some of the safety and ethic issues of using rDNA?
• There will always be concerns regarding the safety of any
new technology, genetic modification and biotechnology
included
- it is virtually impossible to prove that something is entirely
safe under all conceivable conditions
• Organisms in the lab are modified to avoid accidental
release (genes deleted or suicide genes added)
• Genetically modified crops must be safe for consumption
and for the environment
(herbicide resistant crop plants could pollinate
related weed species)
• If genetic screening for disease becomes routine, who
will have access to an individual's genetic information?
