130 terms

Biotech year in review (Cluster 1 and 2)

Production of recombinant insulin requires an expression vector whcih includes promoter of the lac operon regulating beta-galactosidase gene fused to DNA sequence coding either A or B peptide of insulin. The fusion point between mentioned above DNA sequences has:

a. stop codon for beta-galactosidase gene
b. start codon for the insulin sequence part
c. to be in frame between beta-galactosidase gene and sequence coding for the insulin part.
D. a promoter for the insulin part
after 30 cycles of PCR, there is how many copies?
Real-time PCR is a powerful diagnostic tool. Using real time PCR one can measure the original amout of target DNA in the sample by

a. detecting how many cycles it takes to amplify specific amount of target DNA in comparison to standard DNA sample with known DNA concentration
b. measuring final yield of amplified target DNA in comparison to standard DNA sample with known DNA concentration
c. running electrophoresis and measuring the intesity of the DNA band containing target amlicon
To determine DNA sequence using Sanger, one needs to

a. perform 4 independant chemical reactions with the same DNA fragment in each case cleaving DNA at on of four nucleotides
b. design a DNA primer which will block DNA plymerase at one of four nucleotides
c. run four independent DNA polymerase reactions each containing one of four dideoxyribonucleotides.
d. fragment chromosomal DNA into pieces so that enormous chromosomal DNA size does not interfere with sequencing reaction
restriction endonucleases
these are naturally occuring enzymes protecting host bacterial cells from foreign DNA and cut at palindromic sequences.
restriction endonucleases
ATCGAT is a palindromic sequence that would be cut by _____ _____.
reverse transcriptase
c-DNA libraries are used during new drug design projects. Creation of c-DNA library requires the action of this enzyme to convert mature mRNA sequences into DNA sequences
several genes or a part of a gene
human genomic DNA sequences can contain how many genes?
Promoter of a gene is found in this biological process.
true or false: promoter of a gene contains ATG start codon.
True or false: transcription of the gene terminates at one of three stop codons
frame-shift mutation
single nucleotide insertion in the protein coding region of the gene cause this type of mutation.
the 5' cap and 3' poly A tail is added after this process
circular DNA molecule containing replication origin and often used as a vector in biotech drugs.
number of total codons in the human genome
number of codons that code for the 20 amino acids.
translation is initiated by ribosomes starting at this sequence in eukaryotes.
translation is initiated by ribosomes starting at this sequence in prokaryotes.
gene expression
transcription, translation, transport of mRNA from the nucleus to the cytoplasm, and mRNA degradation rates all effect the rate of this biologic process
a foreign gene placed in a developing animal blastocyst for biotech pharming
cut DNA from an exposed end of DNA; facilitate repair.
an enzyme that cuts the bonds within a polynucelotide chain
this seals up the bonds of a recombinant protein after the desired gene as been inserted
transformation techniques
heat shock, CA, Rb; Electroporation, Transfection
genomic library
A set of thousands of DNA segments from a genome, each carried by a plasmid, phage, or other cloning vector.
cDNA library
A gene library containing clones that carry complementary DNA (cDNA) inserts. The library includes only the genes that were transcribed in the cells whose mRNA was isolated to make the cDNA.
northern blotting
This blotting technique involves RNA
southern blotting
this blotting technique involves DNA
Western Blotting
this blotting technique involves proteins.
1. Denature protein at 95 C
2. Uaw primers (DNA polymerase requires Primers not RNA).
3. 65 C the DNA begins to anneal
4. 72 C the TAC polymerase will begin to occur and extend the primers in each direction.
5. Repeat the cycle, now at 60 C newannealing will occur and you get 4 templates instead of 2.
6. Large amplification reaction
Geometrical progression
this type of progression is something changed by a factor each time
arithmetical progression
something changed by plus 1
advantages of this cloning and analytical process are that it is very sensitive, relatively fast, and produces large amounts of DNA
disadvantages of this cloning and analytical process are that it is very sensitive and not as good with longer DNA molecules. Cannot amplify the whole chromosome or genome. There is a limit to how long the DNA can be to amplify.
real-time PCR
laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.
In the PCR the amplified region is defined by the chose _____ pair.
PCR is a useful tool in _____ practices.
Sanger Sequencing
Dideoxynucleotides halt dna polymerization at each base generating sequences of various lenths that encompass the entire orginial sequence. Terminated fragments are electrophoresed andthe original sequence can be deduced.
point mutation
mutation that affects a single nucleotide, usually by substituting one nucleotide for another
Single nucleotide polymorphism abbreviation. Refers to nucleotide substitutions in a gene from different individuals of the same species.
Sanger sequencing and other sequencing methods are ultimate proof of these genetic changes.
missense mutation
The most common type of mutation, a base-pair substitution in which the new codon makes sense in that it still codes for an amino acid.
silent mutation
A mutation that changes a single nucleotide, but does not change the amino acid created.
nonsense mutation
A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein.
ex vivo
therapy where patients cells are removed, grown in culture, incubated with viral vector to introduce genes and transplanted back into patient
in vivo
The natural conditions in which organisms reside. Refers to biological processes that take place within a living organism or cell under normal conditions
a short single stranded nucleic acid polymer. These are most commonly used to intervate in gene transcription, translation DNA repair, recombination, gene construction and different diagnostic analyses.
Folded RNAs that code for a particular protein; also can start and stop production of the protein
These are nucleic acid ligand binders. Th RNA and DNA can fold like proteins into proteins into defined 3d structurs. They can bind nucleic acid, proteins, small organic compounds of interest.
Enzyme-linked immunosorbent Assay. Proteins bound to the plastic microtiter dish, and then the well is treated with specific antibody linked to an enzyme generating a colored product. This is used to quantify antigen
L- amino acid
all proteins in the human body are made of this type of amino acid. not R-amino acid
This poly ethanol structure is used to stabilize proteins in solution so that they properly fold.
This type of molecule has a lipophilic body with a hydrophilic head
Svedburg units
Units of sedimentation
chemical instabilities
these types of instabilities consist of oxidation, hydrolysis of amide side chain, and hydrolysis of the peptide bond.
physical instabilities
In these instabilities there is no actual change in chemical bonds. They consist of denaturation, aggregation, precipitation, and adhesion to surfaces.
A test for the presence of a substance using the reaction of an antibody to its antigen, making use of the high selectivity of components of biological immune systems.
The movement of suspended particles through a fluid or gel under the action of an electromotive force applied to electrodes in contact with the suspension.
Type of analysis used to separate proteins based on mass. SDS binds to the proteins, giving them a negative charge cancelling the effect of charges from the individual amino acids. Lighter proteins travel faster than heavier ones.
isoelectric focusing
Type of chromatography used to separate proteins based on charge. Stationary gel has an established pH gradient and the mobile phase proteins will travel to the point where the pH equals their isoelectric point.
size exclusion chromatography
Small beads of polymerized glucose, agarose, or acrylamide are manufactured with different sizes of poe depending on the degree of cross-linking of the polymer.. In this the larger particles come out first
ion exchange chromatography
proteins have charges due to amino acid side groups, bind to charged column matrix depending on their charge at a particular pH, anionic:negatively charged matrix(Heparin Sepharose), cationic:positively charged matrix(Q sepharose), elute bound proteins based on charge__then diplace by salt or pH change
reverse phase chromatography
Column has hydrophobic groups on the side and pulls out particles based on their hydrophobic properties. High ionic strength buffers are needed to get the solution out of this chromatography
affinity chromatography
Chromatography method which takes advantage of a proteins affinity to a specific ligand. The most powerful separation technique for proteins. Could be a specific ligand in the chromatography column or could be an antibody and should have a specificity and it is hard to get antigen off of antibodies.
Mass Spectrometry
destroys the compound, use high speed beam of electrons to ionize sample (eject an electron), a particle accelerator put charged particles in flight, a magnetic field deflect the accelerated cationic fragments and a detector records the number of particles of each mass that exit deflector area
transgenic animals
Animals that contain genes transferred from other animals, usually from a different species
knockout mice
genetically engineered mice in which a specific gene has been inactivated, for an example by introducing a deletion in its DNA.
stirred tank, airlift, microcarrier, membrane bioreactor
4 different types of bioreactors
stirred tank
Bioreactor contains a device that acts like a ceiling fan in the medium
bioreactor that the solution is kept moving by air bubbling through the solution
the bioreactor that involves encapsulated cellsin carriers that are in the medium and this allows for continual washing with new medium
membrane bioreactors
This bioreactor is literally a semi-permeable membrane between cell culture and fresh medium. Allows for exchange of nutrients, oxygen, and waste, but does not allow for the cells to cross to the other side.
This type of growth medium consists of putting all the ingredients together, mix it up, and let it grow, without adding anything new to it other than oxygen. No more added nutrients or removal of excrements .
Fed Batch
This type of growth medium consists of a medium similar to batch, but in this one we are going to continue to add new medium until you reach a point when the culture reaches its maximum.
Continuous production
This type of growth medium is the most preferable which is not easy to do using a stir reactor, but if you use a semipermiable membrane you continue to wash, continue to keep it growing. This is very useful when you culture is actually secreting the protein.
Hybrid cell lines that make monoclonal antibodies of defined specificity. They are formed by fusing a specific antibody-producing B lymphocyte with a myeloma cell that grows in tissue culture and does not make any immunoglobulin chains of its own.
fetal calf serum
This is the magical elixir. Mammalian cells do not culture well until you add this.
host-related contaminants
Contaminants native to the cells themselves (examples: viruses, glycosylation variants, n and c terminal variants, and endotoxins)
product-related contaminants
Contaminants that are manipulated end products. Denatured, aggregated, fragmented, or misfolded forms of the produced protein.
process-related contaminants
Contaminants from the media or added substances in purification (examples: media components, fetal calf serum, purification reagents, metals, column materials).
Contaminant removals
Can be done by inactivation (heat, UV, dehydration, solvents, detergens or antibodies) or removal (chromatography, filtration, or selective precipitation methods)...
downstream process
Particulate removal, concentration, protein capture/initial purification, intermediate purification, final purification, and sterilization and formulation.
design stage
This stage of downstream processing starts with samll amounts of the protein in the laboratory setting trying to make a purification process
Scale-up stage
This stage of the downstream process involves applying the design stage to larger quantities of protein.
Separation technique used to separate particles according to mass, shape and density. Greater mass and density settle near the bottom while lighter compounds remain on top. This is meant to simulate a gravitational pull.
salting out
the fractionation method that often follows differential centrifugation. Adding salt to the crude extract causes some proteins to be less soluble and be released
stationary phase
on the resin of the chromatography
mobile phase
solution passing through the column of the chromatography process
adsorption chromatography
Chromatography with a polar stationary phase. Protein of interest binds to polar stationary phase and then released by converting the polar phase to non-polar. hydrophobic interaction or reverse phase chromatography
n-terminus heterogeneity
Indicates the protein has undergone degradation during purification steps. This can be done by endopeptidase or exopeptidases. If done by exopeptidases then it will have this type of heterogeneity.
both the N and C terminal ends of a protein can undergo degradation by _____. These are located in the hepatocytes
inclusion bodies
the most abundant of these are the storage granules that hold the cell's reserve supplies of nutrients. some species of bacteria store carbon, some poly-beta-hydroxyalkanes (carbon in granules composed of lipids), polyphosphate (phosphate...essential component for ATP). These form if the protein is produced in bacterial host cells and may result in denaturation of the protein products.
these methods of preparation strive for this. They include aseptic technique, autoclaves for equipment, filtration of product, gloves, masks, and coats.
This type of contaminant is a bacterial endotoxins. Lipopolysaccharides. Highly charged and have hydrophobic regions. Removed using gel filtration or ion exchange chromatography methods.
formulation stage
at what stage is it difficult to remove viral or viral particles from a solution. This is why you remove them early in the purification process.
any more or less inert substance added to a prescription in order to confer a suitable consistency or form to the drug; a vehicle; inactive ingredients
solubility enhancers
these excipients are needed depending on how many hydrophylic side chains are exposed. Include salts, buffers, detergents or surfactants
anti-aggregation agents
albumin or other inert proteins, detergents or surfactants. Excipients that help prevent proteins binding to surfaces or other proteins
Include ascorbic acid, sodium formaldehyde sulfoxylate, GSH. THey prevent the oxidation of S atoms.
glutamic acid, cysteine and glycine
GSH is composed of these amino acids
used for multi-entry or multi-dose vials. Phenylmurcuric nitrate, thimerosol, beta-hydroxybenzoic acid, phenols, and small alcohols
agent that slows or stops the growth of bacteria
capable of killing bacteria. Antibiotics, antiseptics, and disinfectants can be bactericidal.
osmotic agents
substance that assist in hydrating and stabilizing proteins in solution. Salts, mono and di-saccharides, polyalcohols such as PEG.
Freeze drying takes into effect this chemistry process that bypasses the liquid phase
Freeze drying
Freezing, primary drying, secondary drying. Skips the liquid phase
1st step in freeze drying process. Need to worry about excipients crystallizing at different temperatures and thus causing possible instabilities to the protein
Primary drying
This is the second step of the freeze drying process. Below freezing temperature but the pressure is lowered drastically. As you are pulling of water molecules, what happens to the surface left behind? The evaporation of the sweating as the water molecules enter the gaseous state and the protein will decrease in temperature. So to maintain this temperature we gotta add a tiny bit of heat while lowering the pressure.
secondary drying
Last step of freeze drying. Final water content is brought to 1.0%. Brown sample means impurity. Need to start over
absorption enhancers
Often times starch. So if you have a hydrophilic drug and want to deliver it transdermally you need to encapsulate it with a lipophilic sphere and get it to go through the skin and then break down once in circulation.
Can encapsulate the proteins in biodegradable microsphers.
This long strand of ethanol is bound to proteins to extend half life
This recombinant protein can be formulated with metal ions especially zinc to form stable suspensions
open loop delivery
drug can be delivered at a fixed rate over time. Pulse rate, response rate, general rate. there is no direct feedback. We are either preprogramming a certain rate or manually giving more when we feel we need more. No feedback from blood levels
alzet minipump
this pump is an open loop drug delivery system for insulin
biodegradable polymer implants involve impregnating a ____ with the drug and letting it dissolve in the skin.
closed loop systems
Needs direct feedback. These are insulin pumps taht would need a direct feedback loop to adjust the dose in the patient.
antibody acts as a homing divice and has a toxin or radionuclide attached to it to do damage to specific cells
colloidal particulate carrier systems
consist of liposomes, nanospheres and microspheres, and LDL molecules
LDL molecules
These colloidal particulate carrier systems encapsulate the drug and are targeted towards the liver. This is because it is a normal cholesterol derivative
on the nano level. These colloidal particulate carrier systems encapsulate the drug.
Fatty layer. These colloidal particulate carrier systems can have a lipophilic drug in its bilayer and a lipophobic drug inside the capsule.
the study of the movement of drugs and their metabolites through the body from absorption to excretion (basically, what the body does to the drug)
What the drug does to the body
allometric scaling
disproportional growht; larger animals require less energy/unit mass vs smaller animals b/c of surface area; when animals double in size, the SA is squared, but the weight is cubed, so the SA cannot support the weight; the proportion is not right; proportion changes when animals change size
kidney filtration
proteins less than 60 kDa goe through the glomerulus. 30kDa undergo extensive filtration.
direct link
This PK/PD model is related to the distribution of the drug. Because of its size, physiochemical properties, ability to stick, etc. the distribution is going to be different.
indirect link
this PK/PD model is related to Receptor effects. Not related to the protein in the vascular system rather it's the effect of the protein actually reaching the receptor.
cell lifespan models
apply to cells in the blood. Some protein drugs specifically cause an increase in levels of blood cells, so measuring the number and half-life of blood cells shows the drug is having its effect.
B-cell tolerance
central tolerance is mediated in bone marrow by binding self-proteins which leads to deletion or inactivation of immature b cells that express IgM that reacts with self
Anyone's grade who is saved by these notecards owes Dan a ______.