Chapter 9- Biotechnology and Recombinant DNA (EXAM 2) Flashcards
The manipulation of living
organisms, or cell components to produce useful products.
Biotechnology
Products made by biotechnology
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
procedures that are used to join together (recombine) DNA segments in vitro
Recombinant DNA (rDNA) technology/genetic engineering
1) Population of cells arising from a single parent cell
2) Processes used to create copies of DNA fragments
clone
The production of exact copies (_______) of a particular gene or DNA sequence using genetic
engineering techniques.
gene cloning; cloning
Describe the process of gene cloning
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
What is the goal of gene cloning?
- either to make copies of the gene
2. or to make protein product of the gene
When copies of the gene are harvested what can they be used for?
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
When the copies of the gene make a protein product, and the desired proteins are harvested; what can they be used for?
- 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
in nature organisms with
characteristics that enhance survival are more likely to
survive.
natural selection
Why are bacteria good subjects to study natural selection?
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.
humans select desirable breeds of animals or strains of plants
artificial selection
provide examples of artificial selection
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)
a tool for biotechnology
mutagens
_______________ can be used to increase the chances of obtaining a
desired strain
- Radiating _________ generated a strain that
produced 1000x penicillin
random mutagenesis (mutagen exposure); fungus
a mutation created at a defined site in a DNA molecule
site directed mutagenesis
Why is site directed mutagenesis useful?
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
Cut DNA at defined positions close to or within their recognition sequences
Restriction enzymes
What is the cutting frequency of restriction enzymes?
typically recognize 4-, 6-, or 8-base sequences
Do restriction enzymes cut the same way each time?
Yes
Some produce _______ ends, others produce ___________ (sticky) ends
blunt; staggered
can be used to join two pieces of DNA with complementary ends
staggered ends
Bacterial source of BamHI
Basicillus amyloliquefaciens
Recognition sequence of BamHI
G|GATCC
GCTAG|G
Bacterial source of EcoRI
Escherichia coli
Recognition sequence of EcoRI
G|AATTC
CTTAA|G
Bacterial source of HaeIII
Haemophilus aegyptius
Recognition sequence of HaeIII
GG|CC
CC|GG
Bacterial source of HindIII
Haemophilus influenzae
Recognition sequence of HindIII
A|AGCTT
TTCGA|A
Recognition sequence of HindIII
A|AGCTT
TTCGA|A
Describe the process of Restriction Enzymes and Recombinant DNA
- restriction enzyme cuts dsDNA at its particular recognition sites
- These cutes produce DNA fragment with two sticky ends
- when two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing
- 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
- The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of recombinant DNA
Compatable cohesive ends
Echo slide 13 (unclear)
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)
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
This modification protects the bacterial host DNA from degradation by its own restriction enzyme • Called \_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_ system
restriction modification
Autonomously-replicating DNA used to carry the desired gene to a new cell
vectors
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)
primary vectors in use. Easy to manipulate
plasmids
accept larger pieces of foreign DNA
viruses
types of viruses used to insert correctives genes into human cells
retroviruses, adenoviruses, and herpes viruses
Are there natural mammalian origins (ORIs)?
no (echo-need clarification slide 15)
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)
Would marker genes that can be phenotypically screened be useful?
Slide 16- Echo
An E.coli plasmid vector used for cloning
Echo slide 17
can replicate in at least two different species
shuttle vectors
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
distinguishing vector self-ligation from insert ligation
slide 19-clarify
A molecular technique that allows for the detection of
successful ligations in vector based cloning
the blue white screen
Describe the blue white screen
- 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.
- 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
- The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant
- 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
- 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.
Enzymatic method to amplify (make multiple
copies) a piece of DNA to detectable levels
polymerase chain reaction
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
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
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.
What is unique about the DNA polymerase used in PCR?
use a Taq polymerase that can withstand high temperatures (Echo slide 24 to clarify)
What is the significance of the temperatures and times chosen for each step?
Echo slide 25 to clarify
Thermocycler
echo slide 26
Robocycler
echo slide 27
How is the amplified product detected?
echo slide 28
How does rtPCR/ qPCR differ from standard PCR?
echo slide 28
what is rt-PCR?
echo slide 28
What are the three phases of PCR? Describe these three phases.
- exponential
- Linear
- plateau
Echo slide 29
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.
What is an alternative method to the Taqman method? Describe this method
SYBR green (echo slide 30)
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.
What primers are used in RT PCR?
echo slide 31
Most bacterial mRNAs have short or no ____________
polyA tails
What are some methods of transformation?
- Electroporation
- Chemical Transformation
- Protoplast fusion
- Gene gun
- Microinjection
define transformation
ways to insert foreign DNA into cells
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.
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
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)
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
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
In chemical transformation, what is the process used for yeast?
Similar method to bacteria except lithium chloride is used
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
takes advantage of the fusion properties of protoplasts
protoplast fusion
fusion rate of protoplasts increases in the presence of ______
PEG
What cell types are protoplast fusion most valuable in?
plant and algal cells
Microscopic gold or tungsten particles (approx. 1 µm) coated with DNA are
propelled into the cells
gene gun/ biolistic transformation
During biolistic transformation, what happens if a gold particle lands in the nuclues?
the genes elute off and may be incorporated in the chromosome
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
Transformation method that can be applied to the widest range of cell and tissue types.
biolistic particle bombardment
uses a glass micropipette to inject DNA into the cell
microinjection
What is microinjection not useful for?
impractical for bacterial and fungal cells.
a collection of DNA fragments of one organism, each carried by a plasmid or
virus and cloned in an appropriate host.
DNA libraries
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
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
What library size (# genome equivalents)
is necessary to ensure that all
sequences of the genome are
represented?
echo slide 39
What is the choice of restriction enzyme in making a genomic library?
echo slide 39
Genome to be stored
in library is cut up with
________ _______
restriction enzyme
is made from
mRNA by reverse
transcriptase
complementary DNA (cDNA)
cDNAs are then cloned into a _______ or ______ ______
plasmid or viral vector
Common for eukaryotic libraries but \_\_\_\_\_\_ \_\_\_\_\_\_ may not be completely reverse transcribed into DNA
long mRNAs
What primer sequence will
allow reverse transcription
of all nuclear eukaryotic
mRNAs?
echo slide 41
Describe the process of making a cDNA library
- a gene composed of exons and introns is transcribed to RNA by RNA polymerase
- Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mRNA
- Isolate mRNA from the cell and add reverse transcriptase
- first strand of DNA is synthesized
- the mRNA is digested by reverse transcriptase
- add DNA polymerase to synthesize second strand of DNA
DNA can be made _________ on a solid matrix using a
DNA synthesis machine
chemically (in a lab)
Two complementary ssDNA molecules can be
synthesized and then hybridized to generate a dsDNA molecule
synthetic DNA
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
How can you identify a clone from a library containing your gene of interest?
colony hybridization
Describe the process of colony hybridization
- Obtain a master plate with colonies of bacteria containing cloned segments of foreign genes
- make a replica of master plate on nitrocellulose filter
- treat filter with detergent (SDS) to lyse bacteria
- treat filter with sodium hydroxide (NaOH) to separate DNA into single strands
- add radioactively labeled probes
- probe will hybridize with desired gene from bacterial cells
- Wash filter to remove unbound probe and expose filter to X-ray film
- compare developed film with replica of master plate to identify colonies containing gene of interest
Can you name an alternative method that is available to identify a specific clone in a library?
echo slide 43
How can PCR be used to obtain a genomic or cDNA clone?
echo slide 44
Which cell types are used to make gene products?
bacteria (E.coli)
yeast (S. cerevisiae, plant cells and whole plants, and mammalian cells
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)
What alternative bacteria is more likely to secrete?
echo slide 45
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
May express eukaryotic genes
easily
Low risk of product
contamination by mammalian
pathogens
Large scale low-cost production
plant cells and whole plants
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
What are some therapeutic applications of rDNA?
human enzymes
subunit vaccines
DNA vaccines
gene therapy
examples of human enzymes made by rDNA
human insulin
- human hormone somatostatin (5 mg = 50,000
sheep brains or 8 L bacterial culture)
specific protein from a pathogen (purified from yeast or expressed as a viral surface protein)
subunit vaccines
injection of plasmids carrying genes for a specific
pathogens antigens. The expressed protein then produces an immunological response
DNA vaccines
replace defective or missing genes
gene therapy
an early commercial success of rDNA technology (1978)
human insulin production
Human insulin subunits are ____ and ____ amino acids
21 and 30
Each subunit in the human insulin was made ________
synthetically
Describe the process of making a DNA vaccine?
- gene for an immunogen
- insert gene into an expression plasmid
- transform bacterial cells, grow bacteria, and purify the plasmid DNA
- Immunize with immunogen-expressing plasmid
What is the first approach to gene therapy?
replace or augment a faulty gene with a normal one (First used in 1990)
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
most gene deliveries are performed by what?
most gene deliveries are performed by adeno- and retroviruses, plasmid vectors and other strategies are being tried
which of the gene therapy vectors is most promising?
adeno associated virus
vectors are showing promise
What is the second approach to gene therapy?
A second approach is to inhibit expression of the defective gene by RNA interference
a eukaryotic biological process in which RNA molecules inhibit gene expression
RNA interference (RNAi)
what are the two small RNA molecules that are central to RNAi?
microRNA (miRNA) and small/ short interfereing RNA (siRNA)
RNAi is also called what?
gene silencing
How are the pathways similar and different? How is RNAi useful experimentally?
slide 52
How do you sequence an organism’s genome?
one technique is to random shotgun sequence it
construct a gene library
closure phase
How does the process of random shotgun sequencing work?
- isolate dna
- fragment dna with restriction enzymes
- clone dna in a bacterial artificial chromosome (BAC)
- sequence dna fragments
- assemble sequences
- edit sequences; fill in gaps
How are the gaps determined?
slide 54
_______________: • 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
the science of understanding the function of
genes through computer assisted analysis
bioinformatics
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
What is proteomics?
slide 57
-starts with a phenotype and identifies the responsible gene
Forward genetics (classical genetics):
-starts with a particular gene and assays the effect of its disruption
Reverse genetics:
• 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
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
What is the benefit of using bacteria to produce nanospheres?
drug
targeting and
delivery
• 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
Ti plasmid also carries
genes of opine ________
catabolism
In a plant cell, only a _________ strand enters the plant cell
ssT-DNA
describe a normal infection by agrobacterium
slide 64
What can the Ti plasmid be used for in plants?
Using the Ti plasmid as a vector for
genetic modification in plants
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
Describe the process of using the Ti plasmid as a vector in GMO plants
slide 65
• 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
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
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?
