Recombinant DNA Technology Flashcards
Where are ribosomes made?
Nucleolus
What are three technological advances that have allowed for recombinant DNA cloning?
- Characterization of the enzymes
- Methods for sequencing DNA
- Synthesis of oligonucleotides
What are restriction endonucleases that are involved in recombinant DNA cloning? What are the differences between Type I, II, and III endonucleases?
- Recognize specific sequences within dsDNA and cleave it in a specific manner
- Type I and III: Cleave at distant sites
- Type II: Recognizes short sequences (4-8 bases) and cleaves within or very near recognition site; recognition sequences have symmetry (palindrome); cleave between 3’-O and phosphate bond
What are produced when endonucleases cleave dsDNA?
- Sticky ends = primed sequence
- Primed sequences are available for recombinant DNA construction
What are some examples of restriction endonucleases?
- EcoRI
- BamHI
- HindIII
- SmaI (produces “blunt” rather than “sticky” ends)
What are the steps involved in constructing recombinant DNA?
- Vector DNA with complementary ends to sticky ends of genomic DNA fragment pair together
- ATP is hydrolyzed to provide energy for T4 DNA ligase to bind 3’-OH and 5’-P ends
What are the steps to DNA cloning in a plasmid vector?
1) Enzymatically insert DNA into plasmid vector containing selectable marker (e.g., ampicillin resistance)
2) Now have recombinant plasmid
3) Mix E. coli (for example) with plasmids in presence of CaCl2; heat, pulse
4) Culture on nutrient agar plates containing ampicillin (cells that do not take up plasmid die on ampicillin plates)
5) The transformed cell that has taken up the recombinant plasmid will survive and the plasmid will replicate
6) Cell will multiply into a colony of cells, each with copies of the same recombinant plasmid
What is the life cycle of a bacteriophage?
1) Adsorption/injection (attach to cell wall and inject T4 DNA contents into cell)
2) Expression of viral early proteins in cell
3) Replication of viral DNA in cell and expression of viral late proteins
4) Assembly of new bacteriophages
5) Lysis of cell and release of new bacteriophages
6) Cycle continues
How are bacteriophages assembled with DNA?
- Have a preassembled head and tail
- Concatomer of phage DNA with region between 2 cos sites of around 49kb
- Nu1 and A proteins promote filling of phage head with DNA between cos sites
- Phage genome now in head
- Phage tail attaches only to filled head
- Virion is complete
Why isolate a gene?
- To derive a protein sequence
- To produce pure protein in large quantities
- To study normal functions of proteins
What is the source of genes for isolation?
- Genomic DNA
- mRNA –> cDNA (reverse transcriptase is enzyme that catalyzes)
What does generation of the “library” mean in gene isolation?
- Collection of DNA fragments propagated in host cells
What does screening the library with a probe refer to in DNA isolation?
- Oligonucleotides complementary for a strand
- Antibodies specific for the protein - find specific gene sequences
What are the steps in cDNA cloning?
1) Hybridize mRNA with oligo-dT primer (adds complementary ssDNA sequence - TTTT (complementary to AAAA in mRNA))
2) Transcribe RNA into cDNA
3) Remove RNA with alkali; Add poly(dG) tail (ssDNA now)
4) Hybridize with oligo-dC primer (CCCC complementary to GGGG in cDNA strand)
5) Synthesize complementary strand
6) Protect cDNA by methylation at EcoRI sites
7) Ligate cDNA to restriction site linkers
8a) Cleave with EcoRI (makes sticky ends)
8b) Cut with EcoRI, remove replaceable region
9) Ligate to lambda phage arms (from lambda bacteriophage DNA - lambda vector arms with sticky ends)
10) Package in vitro to create recombinant lambda virions
11) Infect E. coli (individual clones)
What is an oligonucleotide probe?
- Protein sequence of small portion of factor VIII protein
- Based on partial amino acid sequence
- Mixture of different oligonucleotides
How do you make a membrane hybridization assay with an oligonucleotide probe?
- Start with dsDNA
- Melt and place on a filter (now have bound ssDNA)
- Incubate with labeled DNA (now have hybridized complementary DNAs)
- Wash away labeled DNA that does not hybridize to DNA bound to filter
- Can find which clone has gene of interest
- Perform autoradiography (detects radioactive materials)
How can you detect DNA that is complementary to a probe using a phage cDNA library that has been radiolabeled?
- Individual phage plaques on plate with E. coli lawn (each plaque is lysis by one viral particle)
- Place nitrocellulose filter on plate to pick up pages from each plaque
- Incubate filter in alkaline solution to lyse phages and denature released phage DNA (single-stranded phage DNA bound to filter)
- Hybridize with labeled probe; perform autoradiography
- Signal appears over phage DNA that is complementary to probe
How can you create a genomic library using genomic DNA cloning?
- Have human DNA with different genes of interest
- Cleave with restriction endonucleases
- Now have millions of genomic DNA fragments
- DNA fragments inserted into plasmids using ligase
- Creates recombinant DNA molecules
- Introduce plasmids into bacteria
- Creates genomic library
How can you probe a genomic library?
- Place a disc of absorbent paper onto a petri dish with colonies of bacteria containing recombinant plasmids
- Peel paper from dish to produce replica of colonies
- DNA is now bound to paper, lyse bacteria and denature DNA with alkali
- Radioactively labeled DNA probe is added
- Incubate paper with probe and wash
- Expose paper to photographic film
- Position of desired colonies will be detected by autoradiography
How do you produce recombinant proteins?
- Isolate the gene
- Introduce the gene into an expression vector (bacterial plasmid, bacteriophage)
- Transfection of host cells and selection
- Growth of transfected cells (fermentation or cell culture)
- Purify protein
What is an easy prokaryotic host cell to use in recombinant DNA technology?
E. coli (easy to use, inexpensive)
What is G-CSF in E. coli?
- Produced by E. coli
- Granulocyte colony stimulating factor
What is the purpose of IPTG with reference to G-CSF?
- When IPTG is present, cell is induced and lac z gene induces formation of lac z mRNA
- G-CSF replaces lac z gene (therefore G-CSF mRNA is formed, rather than lac z mRNA)
- When E. coli is transformed, G-CSF mRNA expresses G-CSF protein (because cell is induced)
- If cell is not induced (i.e., no IPTG) then no G-CSF protein is expressed
What are important factors in and useful cells for eukaryotic host cells?
- Glycosylation important for activity
- Yeast cells (e.g., Hep. B surface antigen vaccine)
- Chinese hamster ovary (CHO) cells (e.g., erythropoeitin)
What colony stimulating factors do eukaryotic cells produce?
- G-CSF (granulocyte colony stimulating factor)
- GM-CSF (granulocyte/macrophage colony stimulating factor)
- Used following myelosuppressive chemotherapy
- Each will have different effect on different people due to different individual genomes
What are different ways to form peptides?
- Recombinant techniques (salmon calcitonin, human insulin)
- Extraction from natural sources (porcine insulin, glucagon)
- Chemical synthesis (solution phase - oxytocin nonapeptide; solid phase - somatostatin cyclic tetradecapeptide)
- Semi-synthetic approaches (chemical modification of natural peptides)
What are some problems associated with chemical peptide synthesis?
- Incomplete chemical reactions
- Side reactions with FGs attached to R1 and R2 units
- Additional protection steps necessary
- Racemization - loss of stereochemistry
- Synthesis of long peptides (>100 aa) problematic for chemical synthesis
What are synthetic peptides?
- Therapeutic proteins composed of >50aa (peptides consist of 2-50 aa)
- Around 30 drugs currently in clinical use
What is peptidomimetics?
- Synthetic mimics of natural peptides
- Either peptide-based or non-peptide based
What are some problems with peptide drugs?
- Drug delivery issues (poorly absorbed across membranes, low oral bioavailability)
- Short biological half life
- Stabilizing agents usually have to be incorporated
- Drug stability and storage issues (prone to chemical degradation, denaturation of large protein drugs)
What are some reactions that happen to cause chemical degradation in peptide drugs?
- Oxidation
- Hydrolysis
- Deamidation and isomerization
- Diketopiprazine formation (leads to inactivation of peptide drug)
What needs to be considered with formulating biotech products?
- Microbial considerations (contamination)
- Excipients used in parenteral formulations
- Shelf-life of biologic drugs
- Routes of administration/formulations for targeted delivery
In terms of microbial considerations when making biotech products, how can sterility be controlled?
- Proteins are usually sensitive to heat, gas, sterilization, ionizing irradiation (denature)
- Therefore, biologics must be assembled under aseptic conditions
- Final filtration sterilization (pre-filter, then 0.22um membrane filter to try to filter out bacterial cell bodies)
- Equipment and excipients should be sterilized separately to protein API, then combined
In terms of microbial considerations when making biotech products, how can viral contamination be avoided?
- Recombinant host organisms must be tested for viral contaminants
In terms of microbial considerations when making biotech products, how are pyrogens removed?
- Pyrogens are mainly endotoxin (LPS) from gram negative bacteria, but also viral and fungal pyrogens
- Removed using ion exchange chromatography (negatively charged)
- Reverse-osmosis generated water for injection (endotoxin aggregates and does not pass through)
- Can also use dialysis, charcoal, oxidation, dry heating
What excipients can be used when forming biotech products?
- Solubility enhancers (ionic strength, solution pH, add Arg and Lys to solubilize, surfactants to avoid protein aggregation and precipitation)
- Anti-adsorption/aggregation agents (API adsorbs to hydrophobic surfaces - water/air, water/container wall, aqueous phase/utensil)
- Buffer selection is very important with biologic drugs (pH dependence of protein solubility, physical and chemical solubility)
What is the typical shelf-life of aqueous solution biologics?
- Less than two years
- Stability depends on pH, ionic strength, temperature, excipient stabilizers (preservatives, anti-oxidants, bacteriostatic, osmotic agents)
What is the typical shelf-life of freeze-dried biologics?
- More than two years
- Presence of water promotes physico-chemical degradation, so water is removed by sublimation
- Free-drying requires proper excipients (bulking agents, collapse-temperature modifier, lyoprotectant)
- Dried form is in compact state (tablets) - mostly veterinary applications (compressed growth hormone pellets, sustained release) or applied sub-dermally (compressed air-powered rifles)
Why are oral biologics less effective?
- Poor bioavailability
- Proteins are degraded in GI tract
- Poor permeability, passive GI uptake
- “Lipidized” oral biologic formulations (but side-effects are a concern)
Why is parenteral administration advised?
- Variable residence time and disposition (regional capillary vs. lymphatic) for IV, s.c., im, ip
- Nasal, buccal, rectal, pulmonary, intravaginal rings (need absorption enhancing technology for systemic distribution) (decrease absorption-barrier resistance, prolong delivery, controlled release formulation)
- Also have topical administration (patches), and microneedle patches
