Exam 2 Flashcards
Why won’t eukaryotic genes function in a prokaryotic organism?
Prokaryotes don’t have the machinery to remove introns.
Describe functional eukaryotic mRNA.
Introns removed, polyA tail attached, and 5’ cap.
What is an intron?
Part of a nucleotide that is removed within a strand so exons can be connected.
Aids in mRNA stability, localization, and translation in Eukaryotes.
Untranslated Regions (5’ UTR and 3’ UTR)
What does the 5’ Cap do in mature mRNA in Eukaryotes?
Helps attachment to ribosomes and protects from exonuclease activity.
What does the PolyA tail do in mature mRNA in Eukaryotes?
Helps in export from nucleus and protects mRNA.
What does the codon do in mature mRNA in Eukaryotes?
It’s translated into protein.
What are the steps of cloning eukaryotic sequences?
- Total RNA is harvested
- mRNA is isolated using oligo dT beads
- mRNA is converted to single stranded cDNA by enzyme reverse transcriptase
- Enzyme RNaseH is used to degrade mRNA from mRNA-cDNA hybrid
- DNA polymerase forms a 2nd strand of cDNA
- ds cDNA is cloned into vectors to create library.
- Library is screened to find the gene of interest.
How large of inserts can be carried by plasmids when cloning DNA?
10 kb or 10,000 bp
-For library, larger pieces of DNA are often needed. Higher capacity vectors were made.
High Capacity Vectors
Bacteriophage Lamda, Cosmid, Bacteriophage P1, Artificial Chromosomes (BAC, YAC, HAC)
E Coli virus that can carry 10-20 kb of DNA
Bacteriophage Lamda
-DNA is expressed in host cells after infection.
E Coli virus that can carry 34-45 kb of DNA
Cosmids
- combine properties of bacteriophage and plasmids
- after infection, the linear DNA becomes circular in a host cell and replicates as a plasmid
Non-viral nucleic acid transfer usually referring to animal cells.
Transfection
Non-viral nucleic acid transfer usually referring to bacteria, fungi, algae, and plants (non-animal eukaryotes)
Transformation
How can DNA get past the cell wall/membrane?
- Heat Shock
- Electroporation
- Conjugation
Name the steps of Heat Shock.
- Treat with CaCL2 and cool
- Transfer to 40* C
- Pores swell allowing DNA to enter
- Cells are cooled and pores close
- Efficiency is 1 in 1000
Name the steps of Electroporation
- Cells are subjected to high voltage electric field.
- Pores form when dielectric strength is surpassed.
Name the steps of Conjugation
- Uses a bacteria’s natural ability to transport plasmids.
- Conjugative plasmid from helper cell moves a mobilizable plasmid from a donor cell to a recipient cell.
Short single stranded DNA or RNA molecule
Oligo
What are oligos used for?
- Assemble whole genes
- Amplify sequences
- Introduce mutations
- Screen libraries
- Sequence DNA
- Cloning
A normal nucleotide with protection groups added to its reactive groups.
Phosphoramidite
Yield in Chemical Synthesis of DNA
- Purity is a concern
- Usually need 98% coupling efficiency
- Yield=(coupling efficiency^(#bp-1)) x 100
Probability that two nucleotides will form a covalent bond during reaction.
Coupling Efficiency
Name the uses of synthesized oligos.
- Makes probes for genomic library screening
- PCR Primers
- Microarrays
- Linkers/Adapters to introduce RE sites for cloning
Adds RE site to unclonable sequences.
Linkers
Adds novel RE sites.
Adapters
How can long DNA fragments be made?
Synthesize shorter DNA fragments that a complementary and fill in the gaps with DNA polymerase.
Enzyme for synthesis of nucleic acid polymers.
DNA Polymerase
When is DNA only truly understood?
When the base pair sequence is known.
What are the Sanger Sequencing steps?
- Primer, ss DNA template, DNA polymerase, and labeled nucleotides are divided into four tubes.
- DNA replication occurs, but a growing chain is terminated when ddNTP is added.
- Products are separated by size using electrophoresis.
- Results are read from gel (250-350 bands) using UV light or autoradiography. Fragment at the bottom is smallest and the 5’ end of the original DNA
What are the Automated Sequencing steps?
- Four unique dyes are used (one for each ddNTP where N=A,T,G,or C)
- All carried out in one tube.
- Argon laser excites dye and each ddNTP produces a unique color.
- About 500 bp per run, 20 kb per hour.
Enables generation of large quantities of a specific DNA sequence in vitro.
Polymerase Chain Reaction
- revolutionized molecular biology
- developed by Kary Mullis in 1986
- can copy DNA billions of times in hours
List the components of a Polymerase Chain Reaction (PCR).
- 2 synthetic oligo primers (~20 nt)
- Target DNA sequence (template)
- Thermostable DNA polymerase
- All 4 ddNTPs
List the steps in a PCR experiment.
- Components mixed in specific ratios
- Rxn volume is 50 microL.
- Rxn Cycle
1. Denaturation - temp raised to 95C and template strands melt
2. Renaturation/Annealing - temp lowered to 55C and primer anneals to template
3. Elongation - temp raised to 70*C and polymerase synthesized DNA starting at 3’ end of primers at 1 min/kb - New strand serves as template for next cycle
- Run 25-35 cycles
- No purification needed
This adds 1000 bp in less than 10 seconds and is isolated from thermophillic bacterium Thermus aquaticus
Taq Polymerase
-Laroche bought patent from Cetus for $330 MM and has mode over $2MMM.
Name the PCR Applications
- Amplification for detection
- Amplification for cloning
- Gene synthesis
- Sequencing
- DNA finger printing
DNA sequence in prokaryotes located upstream that directs the binding of RNA polymerase so transcription can begin.
Promoter
- Overexpression os a gene can be an energy drain on a cell and inhibit its metabolism.
- It is desirable to regulate promoter activity
Name the components of expression regulation in prokaryotes.
- Operator - region upstream which a repressor or activator binds
- Repressor - protein that binds to an operator or promoter preventing transcription by stopping the binding of RNA polymerase
- Activator - protein that binds to an operator and enhances the rate of transcription
What are the steps of negative gene regulation?
- Repressor binds to operator
- Ligand binds to repressor
- Repressor dissociates
- RNA polymerase free to bind
- Transcription occurs
What are the steps of positive gene regulation?
- Transcription factor recruits RNA polymerase
- Activator binds TF upstream of the gene
- RNA polymerase free to bind
- Transcription occurs
How can gene regulation be controlled?
- Ligand concentration helps control gene regulation/transcription
- Promoters can be induced by temp and environment
- Choice of ligand or promoter effects when the gene will be transcribed
Name characteristics of Protein Production in Eukaryotic Hosts.
- Can perform post translational modification (PTM, i.e. folding/cutting)
- systems may be yeast, insect, mammalian
- no system is ideal since PTM varies among species
What must we consider when choosing a specific eukaryotic system?
- Quality of recombinant protein needed
- Yield needed (higher in simpler cells)
- Ease of cell culture
- Cost of production and purification
Why is Saccharomyces Cerevisiae a common host cell for cloned eukaryotic genes?
- Unicellular
- Genetically well-defined
- Easy to grow
- Contains several strong promoters
- Many PTMs
- Protein products easily purified
- GRAS (generally regarded as safe)
Name 3 S. Cerevisiae expression vectors.
- YEps (Yeast Episomal Plasmids)
- 2 micron plasmid often used as a cloning vector
- Selection done with Auxotrophic strains which can’t produce a particular AA or nucleic acid
- Gal-1-P only active when cells are gown in galactose - YIps (Integrating Vectors)
- Yield is lower than plasmid system
- Linearized to facilitate integration - Yeast Artificial Chromosomes
- Clones a large segment of DNA (~100 kb)
- Not commercially common right now
What other yeasts have been used as expression systems besides S. Cerevisiae?
- Pichia Pastoris
- Higher cell density (more product)
- Easier purification
- Produced more than 100 active proteins - Hansenula Polymorpha - produces functional hemoglobin A
- Schizosaccaromyces Pombe - expressed multiple human genes
- Candida Utillis - produces sweetener monellin
Steps of PCR-Amplified Oligo-Directed Mutagenesis
- Target gene cloned into plasmid vector
- Sample split into two aliquots (tube)
- PCR primers added to each (one complementary, one is a mismatch)
- Primers anneal differently in each tube
- Linear products are mixed, denatured, and renatured.
- Renaturation forms circular strands with nicks that are repaired in vivo.
- All plasmids contain “error”
A bacteriophage vector produce M13 virus particles that contain mutated DNA, but only 1-5% contain target mutation.
M13 DNA Oligo Directed Mutagenesis
-Very complex procedure
How is plasmid DNA oligo directed mutagenesis different?
A plasmid is used rather than a virus. Plasmids have multiple cloning sites and multiple selectable markers. Oligos are used to introduce mutations and the markers are for screenings. About 90% have mutation of interest.
When is random mutagenesis needed?
When it is not clear which AA changes give a desired performance. Must generate all possible changes and screen for desired performance.
What are the advantages of random mutagenesis?
- Detailed information of AA role is not needed.
- Unexpected mutants can be made from other AA changes, some useful.
Name the random mutagenesis methods.
- Degenerate Oligo Primers
- Nucleotide Analogues
- Error Prone PCR
- DNA Shuffling - 2 DNA sequences with 3 cut sites will produce 14 possible hybrids
- Synthetic Amino Acids - expands on 20 naturally occurring AAs
- Must synthesize a novel tRNA and novel tRNA synthesase
Why are protein engineering strategies in effect?
Of the thousands of enzymes that have been studied/characterized, about 20 make up 90% of usage in industrial biotech processes.
-Most naturally can’t survive in industrially relevant rxn conditions. (i.e. High Temps or exposure to organic solvents)
The enhancement of thermostability by addition of cysteine residues to the native T4 lysozyme is done by what process?
Adding Disulfide Bonds
- If T4 was modified to add cysteine in groups 2,4 and 6, this would add 1, 2, or 3 disulfide bonds.
- Certain bonds cause activity loss (21-142), but we want minimum loss.
At high temperatures, asparagine undergoes deamination rxn and forms aspartic acid. How is this avoided?
Change asparagine to other amino acids.
Changing one increased stability.
Changing two increased it more.
Changing to aspartic acid lowered it.
How does Ala-51 and Pro-51 change binding affinity, catalytic rate constant, and catalytic efficiency over Thr-51?
Binding Affinity - increase 2 fold with Ala, 100 fold with Pro
Catalytic Rate Constant - Decreased in both cases
Catalytic efficiency - increased in both cases
Why have Molecular Diagnostics been used and what methodology is used?
It is needed for the rapid identification of viruses, bacteria, fungi, parasites, proteins, and small molecules to prevent, control, and treat problems. Immunological or DNA detection methodologies have been devised.
What makes successful Molecular Diagnostics?
Specificity, Simplicity, and Sensitiviy
Name two Immunological Diagnostic Procedures.
ELISA and monoclonal antibodies
-IDP takes advantage of immune response by antibody/antigen binding which is highly specific.
Has been used to detect antibody concentrations for HIV and West Nile Virus, and food allergens like milk, peanuts, and eggs.
Enzyme Linked Immunosorbant Assay (ELISA)
What are the steps of ELISA?
- Sample is immobilized on a 96 well microteter plate.
- Add a primary antibody which targets the antigen and wash.
- Add a secondary antibody which binds only to the primary one.
- Add a colorless substrate that reacts with secondary antibody.
- Measure color/fluorescence produced.
- Compare with control.
Why are monoclonal antibodies used over polyclonal?
Monoclonal antibodies target a specific antigen determinant (epitope) on the antigen. They can be grown in cultures as opposed to in animals like rabbits.
How are monoclonal antibodies made?
- Mice exposed to antigen.
- Mice tested with ELISA to determine if response occurred.
- Mice killed and spleen ground up to release B-cells.
- Cells mixed with myeloma cells (cancerous B-cells)
- Treat to induce fusions
- Use selectable markers to identify hybridomas.
- Immunoassay to identify antibody production.
- Dilute until monoclonal
Name two advantages of monoclonal antibodies.
- Can be maintained indefinitely in culture.
2. hybridoma cells make ELISAs much more specific and sensitive.
Why are DNA diagnostic systems useful?
We can screen for a specific nucleotide sequence to diagnose a potential pathogen or disease.
Give the steps of hydrogen bonding DNA diagnostics.
- Bind ss (single strand) DNA (target) to a membrane support
- Hybridize with labeled ss DNA (probe)
- Wash away unbound probes
- Detect hybridized sequences using label
(target can be amplified using PCR)
