Applied Molecular Biology Flashcards
Mutations resulting in a change of restriction sites in a gene can be used to perform pedigree analysis of individuals. In other words, the same gene of two individuals may be cleaved differently by a given restriction enzyme and result in gene fragments of different size. This type of difference is called what?
Restriction Fragment Length Polymorphism (RFLP)
If RFLP occurs as a result of mutation in DNA coding for an essential protein, the RFLP can be used to do what? If it occurs in gene loci that have no known function or not related to disease, it can be used to do what?
diagnose the disease; identify the individual (forensic pathology)
What is the molecular basis for Sickle Cell Anemia?
substitution of T and A in the template strand of the sixth codon for beta-globulin changes the codon from glutamic acid to valine
What physical properties of hemoglobin are effected in SCA?
at reduced oxygen tension, the hemoglobin precipitates
What is the basis of detection for SCA?
loss of restriction enzyme site (MstII) - when this gene is amplified (PCR) and subjected to treatment with MsII, the pieces of DNA generated are different from that in a normal individual
If the mutation is known, one can rapidly determine genotype of patient by hybridization of ____ ____ oligonucleotides (labeled with 32P) to PCR amplified region of gene in question
alley specific oligonucleotides (ASOs)
The basis of DNA finger printing is RFLP. Because some mutations some segments of DNAs from different individuals contain different restriction enzyme sites and thus these segments would yield what?
different sized fragments when digested with enzymes
Polymorphism in angiotensinogen is being used to predict ____ in cardiac patients
hypertension
Certain sections of DNA are known to be hypervariable. What does this mean?
no two individuals (excluding identical twins) possess the exact same restriction sites in this region - can be used to identify individuals
“Gene chips” or ____ are revolutionary new technology for analyzing ____ _____
microarrays; gene expression
In regards to gene chips, hundreds of unique gene fragments (DNA) are fixed to glass slides by robotic work stations used in computer chip assembly. RNA isolated form cells or tissues can be converted to ____ with reverse transcriptase
cDNA
note: synthesis occurs with incorporation of fluorescently labeled nucleotides
In regards to gene chips, _____ of fluorescently labeled cDNA to DNA ‘spots’ on the chip - each of which corresponds to a unique gene-allows quantitative measurement of message
hybridization
In regards to gene chips, if two different cell populations are carried through the procedure and each cDNA pool is labeled with a different florescent nucleotide, then a differential expression level will result as the computer sums data analyzed in two different channels. True or false?
true
What are the major problems of gene therapy?
efficiency of integration and expression of therapeutic genes
In regards to gene therapy, in many cases only about 5-10% of normal expression may be enough to bring about a correction of the biochemical deficiency. True or false?
true
There are both viral and non-viral gene delivery strategies. The non-viral suffer from low efficiency of ____ into cells. The viral have problems like inducing an ___ ____ which will nullify the therapy or cause cancer
delivery; immune response
What is the advantage and disadvantage of the retrovirus method of gene delivery?
advantage: stable chromosomal integration; easy to produce
disadvantage: transduces dividing cells only; risk of insertional mutagenesis
What is the advantage and disadvantage of the adenovirus method of gene delivery?
advantage: high titers achievable; easy to produce
disadvantage: transient expression induces immune response
What is the advantage and disadvantage of the adeno-associated viruses method of gene delivery?
advantage: nonpathogenic; broad target range; site-specific integration
disadvantage: difficult to produce; carries only small genes
What is the advantage and disadvantage of the transfection method of gene delivery?
advantage: easy to prepare
disadvantage: no well documented role in vivo
What is the advantage and disadvantage of the gene gun method of gene delivery?
advantage: no viral sequences
disadvantage: transient expression; variable transduction efficiency
What is the advantage and disadvantage of the direct injection method of gene delivery?
advantage: useful in vivo
disadvantage: low transduction efficiency
What is the advantage and disadvantage of the cationic liposomes method of gene delivery?
advantage: easy to prepare
disadvantage: transient and less efficient transduction than viral vectors
In regards to viral vectors, ____ are good gene therapy vectors because they infect cells with high efficiency and integrate the therapeutic gene into the genome of the host cell.
retroviruses
One drawback of retroviruses is that they only infect ____ cells.
dividing
In regards to viral vectors, ____ vectors are produced which can package a therapeutic gene but which are replication deficient
adenoviral
The major advantage of adenoviral vectors is that they can infect what cells? One major limitation is transient expression due to the fact that the adenoviral DNA is not what?
advantage: they can infect dividing or non-dividing cells
limitation: adenoviral DNA is not integrated into genome of the host cell
In regards to viral vectors, adeno-associated virus vector integrates into the genome at a defined site, is non-pathogenic, and can infect non-dividing cells; however, they lack a good ____ cell line and can only incorporate ___ DNA fragments in them
packaging; small
Non-viral methods all suck. True or false?
true - all suffer from low efficiency of DNA uptake cells
Retrovirus enters a human cell through recognition of distinct receptor proteins on the cell’s surface, it uses its own ____ ____ to make a cDNA copy of its genome (its now a provirus), this is incorporated into the host’s ____ where this viral dNA directs formation of its RNA plus protein products
revers transcriptase; genome
Therapeutic gene replaces ____ ___ protein genes, however helper virus in packaging cell supplies coat proteins to form “____ vector” virus
retrovirus coat; safe
In regards to the cloning strategy, most of the genes which code for the viral proteins are removed and replaced by the therapeutic gene. A ___ ___ RNA is also introduced into the cell. It can make viral proteins but cannot be assembled into the new virus
helper viral
In regards to the cloning strategy, the helper viral RNA’s segment for viral proteins can be transcribed and translated. These proteins plus the “_____” viral RNA can be assembled into a ____ vector
engineered; safe
In regards to the cloning strategy, after the safe vector has been incorporated into the human genes, can it form new virus particles?
no
In regards to target cells, transformed ____ containing the gene for human factor 9 have been shown to release their protein products for 2 weeks
fibroblasts
In regards to target cells, ___ cells from a woman donor with familiar hypercholesterolemia have been engineered in vivo to express the ___ receptor
liver; LDL
In regards to target cells, these cells have been engineered to contain genes for beta-globin or adenosine deaminase (to treat SCID)
lymphocytes and bone marrow cells
Recent news: all human therapies of this type have provided only short-term success (months); however long term expression has just been achieved for T-cells and experiments are underway (SCID)
transforming stem cells
In regards to rejection of the cloned protein, if the patient produces absolutely none of the cloned gene protein product, the protein might be immunologically rejected. Therefore, gene therapy will probably work best in what individuals?
individuals having at lease some capacity to make the normal protein
If an introduced retrovirus inserts not the genome near a _____ perhaps it can be activated and cause cancer
protooncogene
Since 1990, a number of patients have undergone adenosine deaminase gene therapy for what?
SCID
Patients with critical limb ischemia (candidate for amputation) have had their limbs spared by injection of plasmid DNA designated ____ directly into muscle
VEGF
note: stimulated angiogenesis helps restore circulation
Patients with _____ have a block in T and NK lymphocyte differentiation due to gammaC cytokine receptor deficiency. Retroviral infection of CD34 T cells showed improvement.
SCID-X1
____ disease is a rare disorder which results in brain degeneration and death by about 10 years of age
Canavan’s
Caravan’s disease is due to an inherited absence of the enzyme ____ which converts N-acetylasparatae to acetate and aspartate. Vital precursors for myelin formation are insufficient without proper levels of this
aspartoacyclase
What are the advantages of plasmid DNA vaccines?
long lived immune response and simultaneous inoculation for multiple disease protection
CGD - lack of phagocytes was treated with what?
adenovirus containing therapeutic gene; 2-3 patients showed clear benefit
Parkinson’s disease was treated with what?
AAV + neuturin gene infusion; 40% of patients showed improvement
To treat melanoma, researchers engineered patient’s lymphocytes with retrovirus and TCR to attack cancer cells better. What was the result?
10-20% improvement
Blindess gene trials Leber’s congenital amaurosis gene therapy in several patients led to modest improvement in ___ function
retinal
Deafness gene therapy in guinea pigs. Adenovirus delivery of gene that caused regrowth of cochlear hair cells. Animals regained ___% of original hearing threshold
80
