Flashcards for Exam 2
starting with "recombinant DNA technology"
What is Plasmid DNA
Small circular pieces of self-replicating DNA found primarily in bacteria.
What is Recombinant Plasmid DNA
A type of plasmid DNA that has been artificially altered to include DNA from another source (such as a gene of interest).
Plasmid traits
- Considered extrachromosomal
- Approx 1 to4 kilobases.
What are vectors
Pieces of DNA that can accept, carry, and replicate other pieces of DNA
What is Calcium Chloride Transformation of Bacterial Cells
a method used to introduce foreign DNA into bacterial cells.
- “inefficient process”
Process of CaCl2 Transformation
- bacterial cells are immersed into a cold CaCl2 solution for 30 minutes.
- plasmid DNA is added, and heat ( 30 secs at 42 C) shock temporarily opens the membrane, allowing DNA to enter.
- plasmid DNA enters bacterial cells and is replicated and expressed in their genes.
What is Electroporation
applying a brief pulse of high voltage electricity to create tiny holes in the bacteria cell wall to allow DNA to enter.
What are the advantages of Electroporation
- rapid and more efficient
- require fewer cells
With reference to recombinant bacteria, what is “selection”
Selection is the process of identifying recombinant bacteria while preventing the growth of non-transformed ones (contain plasmid without foreign DNA)
What is Antibiotic Selection
a method used to identify bacteria that have successfully taken up a plasmid containing a gene for antibiotic resistance
- cannot distinguish between bacteria that have taken up the desired recombinant plasmid and bacteria that have taken up a plasmid that has recircularized without the successfully taking it in.
What is Blue-White Selection
a technique used to identify recombinant bacteria. Bacteria with plasmids containing the lacZ gene can hydrolize X-gal, turning blue. If foreign DNA is inserted into the plasmid, the lacZ gene is disrupted, and these bacteria remain white.
What and when were the first recombinant human proteins marketed
Insulin in 1982 and Growth Hormone in 1985
Technique for insulin
inserting the cloned human insulin cDNA sequence into a plasmid. The plasmid is then introduced into bacterial cells, which are used to synthesize the insulin protein encoded by the cloned gene.
What was the source of growth hormone prior to recombinant technology
human cadavers
Practical Features of DNA Cloning Vector
- Size: Small enough to be separated from the host’s chromosomal DNA.
- Origin of replication (ori): Starting point of DNA replication, allows plasmid to replicate independently.
- Copy number: plasmids per cell; usually low, but have high copy number.
- Multiple cloning site (MCS): Contains recognition sites for several restriction enzymes where DNA inserts are cloned.
- Selectable marker genes: Allow selection of transformed colonies.
- RNA polymerase promoter sequences: Enable transcription in vitro and in vivo.
Why cant you use human insulin genomic DNA for a cloning project
genomic DNA contains introns (non-coding regions), which bacteria cannot process.
What are DNA Libraries
collections of DNA fragments that represent the complete genome of an organism. “held” in plasmid vectors within host bacteria (ex. E. coli)
What are the two types of libraries
- Genomic DNA Libraries
- Complementary DNA libraries cDNA
Genomic libraries:
- Isolate chromosomal DNA from tissue of interest and digest with a restriction enzyme to produce fragments.
- Digest the vector with the same enzyme for compatible sticky ends.
- Use DNA ligase to ligate genomic DNA fragments with plasmids or adapters/linkers.
Disadvantages of Genomic Libraries
- Inclusion of Introns: Both introns and exons are cloned; most genomic DNA in eukaryotes is introns, resulting in a library rich in non-coding DNA.
- Search Difficulty: Large genomes make finding the gene of interest challenging and time-consuming.
- Gene Expression Info: Does not provide data on levels of gene expression.
Making complementary DNA from mRNA
- Extract mRNA from tissue of interest
- Use reverse transcriptase to create double-stranded DNA from mRNA.
- Add short linker DNA sequences with restriction sites to cDNA ends.
- Cut with a restriction enzyme and ligate with a cut vector to create recombinant vectors.
- Transform bacteria with recombinant vectors.
Advantages of cDNA Libraries
- Represents actively expressed genes from the tissue from which mRNA was isolated.
- Does not include introns.
- Can isolate genes expressed under specific conditions in a tissue.
Disadvantages of cDNA Libraries
Difficult to create if the source tissue lacks abundant mRNA.
Library Screening - Process of Colony Hybridization
- Grow bacteria with recombinant DNA on an agar plate.
- Place a nylon or nitrocellulose filter over the plate.
- Treat the filter with an alkaline solution to lyse bacteria and denature DNA; expose to UV light.
- Denatured DNA binds to the filter as single-stranded DNA.
- Incubate the filter with a probe tagged with a radioactive nucleotide or fluorescent dye.
- Probe binds to complementary sequences on the filter (hybridization).
- Wash the filter to remove excess unbound probe.
- Expose the filter to X-ray film or a digital camera to detect the fluorescent probe.
- Compare the film or image to the original agar plate to identify colonies with the gene of interest.
Polymerase Chain Reaction (PCR)
A technique for amplifying target DNA quickly.
Developed in 1983 by Kary Mullis.
PCR Process- What components are needed for a PCR reaction?
Target DNA, nucleotides (dATP, dCTP, dGTP, dTTP), buffer (MgCl₂), DNA polymerase, and forward and reverse primers.
PCR Process- What is the role of the buffer in a PCR reaction?
Maintaining the optimal pH and ionic strength for the DNA polymerase to function effectively.
(Typically contains MgCl2).
PCR Process- What are primers in PCR, and what is their function?
Primers are short single-stranded oligonucleotides (18-22 nucleotides long) that bind to the target DNA to guide DNA polymerase for replication.
PCR Process- How is the PCR reaction initiated after adding all components?
The reaction tube is placed in a thermocycler, which amplifies the target DNA through repeated cycles of heating and cooling. thermocycler for amplification.
PCR Cycle in Thermocylcer - What are the three stages of a PCR cycle?
- Denaturation (94-96°C)
- Annealing (52-58°C)
- Extension (70-75°C)
What happens during the denaturation stage of PCR?
DNA is heated to 94-96°C to break hydrogen bonds between the strands, separating them for replication.
What occurs during the annealing (hybridization) stage in PCR?
Primers hydrogen bond with their complementary sequences at the 3’ ends of the target DNA at 52-58°C.
What happens during the extension (elongation) stage in PCR?
DNA polymerase copies the target DNA at 70-75°C, synthesizing new DNA strands.
What is the result of one PCR cycle?
The amount of target DNA doubles at the end of each cycle.
How many times is the PCR cycle typically repeated?
The PCR cycle is repeated 20-30 times.
Advantage of PCR
- Amplifies millions of copies with a small amount of starting material in a short period of time.
-To calculate # of copies - T x 2^N
N = # of PCR Cycles
T = represents the # of initial target DNAs - quicker and more effective than DNA libraries
What DNA Polymerase is used for PCR
Taq DNA Pol - isolated from a species known as Thermus Aquaticus that thrives in hot springs.
Disadvantage of PCR
Depends on the design of the primers that match the target sequence. Poorly designed primers can lead to non-specific amplification, resulting in unwanted products.
What does Taq Polymerase do
it puts a single adenine nucleotide on the 3’ end of all PCR products
How do researchers use the adenine added by Taq polymerase in PCR products?
Researchers use a T-vector that has single-stranded thymine on each end, allowing it to base pair with the adenine in the PCR products for cloning purposes.
DNA Sequencing - What is the Sanger Method used for?
The Sanger Method, or chain termination sequencing, is used to determine the sequence of nucleotides in a cloned gene.
Who developed the Sanger Method and when?
The Sanger Method was developed by Frederick Sanger and colleagues in 1977.
What components are required for the Sanger sequencing reaction?
The reaction requires a single primer, denatured DNA template, all 4 dNTPs, DNA polymerase, buffer with MgCl₂, and dideoxynucleotides (ddNTPs).
How do dideoxynucleotides (ddNTPs) terminate DNA synthesis in the Sanger Method?
ddNTPs have a 3’ H instead of a 3’ OH, preventing the formation of a phosphodiester bond with the next nucleotide, causing chain termination.
What is the advantage of high-throughput computer-automated sequencing using the Sanger method?
- more than 600 nucleotides can be sequenced per reaction (using capillary electrophoresis)
- essential in completing the Human Genome Project
- uses only 1 reaction tube rather than 4
How are ddNTPs detected in Sanger sequencing with capillary electrophoresis?
ddNTPs are labeled with different fluorescent dyes, and a laser scans DNA fragments in a capillary tube, causing each dye to emit light at different wavelengths.
What is the role of the computer in high-throughput sequencing with capillary electrophoresis?
The computer collects light patterns emitted by the fluorescent dyes and converts them into DNA sequences, handling up to 900 base pairs at a time.
What is next-generation sequencing (NGS) with pyrosequencing?
NGS, such as the Roche 454 system, uses pyrosequencing, where pyrophosphate reactions produce light to sequence DNA fragments attached to beads through emulsion PCR.
How does ATP sulfurylase contribute to pyrosequencing in NGS?
ATP sulfurylase converts pyrophosphate (PPi) into ATP, which is then used in a reaction with luciferase to produce light in a luciferin-luciferase reaction.
What is the role of luciferase in pyrosequencing?
Luciferase converts luciferin into oxyluciferin using ATP, generating visible light that is detected to sequence DNA in pyrosequencing.
What is the principle of NGS (2nd generation sequencing) using the Ion Torrent PGM? Personal Genome Machine
- Utilizes the release of H⁺ ions on a semiconductor
- DNA → Ions → Sequence
- Nucleotides flow sequentially over the ion semiconductor chip
- One sensor per well per sequencing reaction
- Direct detection of natural DNA extension
- Millions of sequencing reactions per chip
What are the key features of 3rd-generation sequencing (3rd GS) using Oxford Nanopore Technologies’ MinION?
- Sensor detects changes in ionic current at nanopore
- nanopore formed in membrane, DNA or RNA pass through
- 10+ kb read lengths
- Error rate ~5%
What is Southern Blotting used for
Used for gene copy number determination, gene mapping, mutation detection, and PCR product confirmation
What are Key Southern Blotting Steps
- Cut DNA with restriction enzymes
- Separate fragments by agarose gel electrophoresis
- Then bascially follow the steps of colony hybridization.
- Using audioradiography or a digital camera, the number of bands will represent gene copy number
What is Northern Blot Analysis used for
To study gene expression by analyzing mRNA
What are the key steps in Northern blot analysis
- Isolate RNA from tissue of interest
- Separate RNA by gel electrophoresis
- Blot RNA onto a nylon filter, UV to fix onto filter.
- Hybridize with a labeled DNA probe
- Detect bands on autoradiograph, showing the presence and size of mRNA
- So it determines whether a gene of interest is expressed ( if binded) as the probe corresponds to it.
How is reverse transcription PCR (RT-PCR) used to study gene expression?
Used to study mRNA levels when detection is below Northern blot sensitivity
What are the key steps in reverse transcription PCR
- Isolate mRNA
- Use reverse transcriptase to make cDNA
- Amplify cDNA region with gene-specific primers using PCR
- Run agarose gel to separate amplified cDNA
- The amount of cDNA produced reflects the amount of mRNA and gene expression levels
How is real-time or quantitative PCR (qPCR) used to study gene expression?
- Quantifies amplification in real time
- Uses special thermal cyclers with lasers to scan light through PCR reactions
- Fluorescence emitted by a probe or DNA-binding dye correlates with the amount of PCR product
- Light is captured by a detector and analyzed to quantify PCR products after each cycle
What are the two approaches to qPCR for studying gene expression?
- Taqman probes:
- Probes are complementary to target cDNA between primers
- Contain a 5’ reporter and a 3’ quencher
- Fluorescent light is emitted when the reporter is excited by the laser and then cleaved by DNA pol, leaving only the quencher, - SYBR Green:
- This dye binds to double-stranded DNA (dsDNA) during the PCR amplification process. When it binds, it fluoresces, allowing for the detection of DNA.
What is Fluorescence in situ hybridization (FISH) used for?
- Used to identify a gene’s location on a chromosome.
- Used to analyze genetic disorders in tissues
- Visualize the presence and localization of specific RNA molecules in tissues
Fluorescence in situ hybridization (FISH) key steps
- Isolate chromosomes on a microscope slide
- Incubate with fluorescently labeled DNA/RNA probe for the gene of interest
- Probe hybridizes to complementary sequences on chromosomes
- Expose the slide to fluorescent light to visualize probe binding
What is a challenge of biotechnology regarding proteins?
Understanding and controlling protein folding.
Characteristics of proteins
- Complex molecules built of chains of amino acids
- Have specific molecular weights
- Have electrical charges
- Hydrophilic - water loving
- Hydrophobic - water hating
Why is protein folding important
The structure and function of a protein depend on proper folding. Incorrectly folded proteins lose their function and can be harmful.
- Can lead to diseases like Alzheimer’s, forms of cancer, and even some heart attacks.
Who described the two regular secondary structures of proteins in 1951?
Pauling and Corey described alpha helices and beta sheets; the structures are fragile and hydrogen bonds are broken easily.
There are four levels of protein structural arrangements -
What is the primary structure of a protein?
The primary structure is the amino acid sequence.
What is the secondary structure of a protein?
The secondary structure occurs when amino acid chains fold or twist due to hydrogen bonds, forming alpha helices or beta sheets.
What are the most common shapes
Alpha Helix and Beta Sheets
How are alpha helices formed?
Alpha helices are right-handed spirals stabilized by hydrogen bonds linking nitrogen and oxygen atoms of different amino acids.
How are beta sheets formed?
Beta sheets are formed by hydrogen bonds linking nitrogen and oxygen atoms, creating parallel or anti-parallel sheets.
What is the tertiary structure of a protein?
the three-dimensional shape formed when alpha and beta cross-link. IT DETERMINES THE PROTEIN’S FUNCTION.
What is the quaternary structure of a protein?
The quaternary structure is a unique, three-dimensional complex formed by several polypeptides.
What is glycosylation?
post-translational modification where carbohydrate units (sugar) are added to specific locations on proteins.
How many post-translational modifications occur within eukaryotic cells?
More than 100 post-translational modifications occur within eukaryotic cells.
What are three ways glycosylation can affect a protein’s activity?
- Increasing its solubility
- Helps proteins correctly position themselves within cell membranes.
- Extending the active life of a molecule in an organism
What is an example of a glycoprotein used in disease treatment?
Glycoproteins can be used as a new way to target and destroy B-lymphoma cancer cells.
How does the treatment using glycoproteins for B-lymphoma work?
The glycoprotein is combined with a nanoparticle loaded with a chemotherapy drug, targeting cancer cells and increasing the effective dose while protecting normal tissues.
What is a time-tested technology that uses proteins in manufacturing?
Brewing, winemaking, and cheese making.
Recombinant DNA technology made it possible to produce what proteins on demand
- Enzymes
- Hormones
- Antibodies
How are target proteins produced in biotechnology?
Proteins are produced via microbial or mammalian cell culture in a complicated and time-consuming process.
What is a bioreactor?
A bioreactor is a cell system that produces biological molecules, typically used to produce large batches of the desired protein.
How are cells stimulated to produce target proteins in bioreactors?
Cells are stimulated through precise culture conditions, including balancing temperature, oxygen, acidity, and other variables.
What happens after the proteins are produced in bioreactors?
The proteins are isolated, tested at every step of purification, and formulated into pharmaceutically active products, while complying with FDA regulations.
How are biosynthetic corneas used in healthcare?
Biosynthetic corneas made from cross-linked recombinant human collagen (produced in yeast cells) help regenerate damaged eye tissue and improve vision.
How are proteins used in screening for diseases?
Proteins, like monoclonal antibodies, are used to detect early biomarkers of diseases. Example: PSA (Prostate-Specific Antigen) test for diagnosing prostate cancer.
What are some industrial applications of proteins?
- Food processing
- Textiles and leather goods
- Detergents
- Bioremediation
What is directed molecular evolution in protein engineering?
a method used to create proteins with new or enhanced properties by mimicking the process of natural evolution in the lab. It involves introducing random mutations into a protein’s gene,by inducing mutagenic PCR.
What is site-directed mutagenesis in protein engineering?
Site-directed mutagenesis involves introducing specific, predefined changes in a specific location of a sequence of a protein.
Give an example of site-directed mutagenesis application.
A company produced bacteria and industrial enzymes that can tolerate high cyanide concentrations, which are normally toxic for most bacteria.
What are the two major phases in protein production?
Upstream processing (protein expression in cells) and downstream processing (protein purification, function verification, and preservation).
What is upstream processing in protein production?
It involves selecting a cell to be used as a protein source and expressing the protein within the cell.
What is downstream processing in protein production?
It involves purifying the protein, verifying its function, and ensuring it is stably preserved.
Why are bacteria commonly used in upstream protein expression?
Bacteria, such as E. coli, grow quickly, are easy to genetically alter, and fermentation processes for bacterial protein production are well understood.
What are inclusion bodies in protein production using E. coli?
they are foreign proteins that accumulate in the cell’s cytoplasm, which must be purified.
What is a fusion protein?
a target protein (the one you want to study or use) is combined with another protein that has special properties, like the ability to stick to a purification column.
What are the advantages of recombinant protein production in E. coli?
- E.coli genetics are well understood
- Nearly unlimited quantities of proteins can be produced
- Fermentation tech is well understood.
What are the disadvantages of recombinant protein production in E. coli?
- Foreign proteins need refolding
- E. coli cannot fold proteins in ways required for many proteins used in mammalian systems
- Some proteins inactive in humans.
How can bacteria be grown for large-scale protein production?
Bacteria can be grown in fermenters (anaerobic) or bioreactors (aerobic).
Why are oxygen levels and temperature monitored in bioreactors?
To ensure ideal conditions for cell growth during protein expression.
What is the role of IPTG in protein expression?
IPTG effectively initiates the expression of genes under the control of the lac operon.
What role do fungi play in protein expression?
Fungi are a source of a wide range of proteins used in products like animal feed and beer, and many species are used as hosts for engineered proteins.
Why are fungi valuable in protein expression?
Fungi are eukaryotic and capable of post-translational modifications, allowing proper folding of proteins.
What types of products are proteins from fungi used in?
Proteins from fungi are used in products like animal feed and beer.
How are plants used in protein expression?
They are genetically engineered to produce specific proteins that do not occur naturally.
What is an advantage of using plants for protein expression?
plants can grow quickly and produce many offspring, this allows for the large-scale production of proteins quickly.
Which plant is a popular choice for biotech protein production, and why?
The tobacco plant is a great choice because it can rapidly produce large quantities of proteins once genetically modified.
What are the disadvantages of using plants for protein expression?
Not all proteins can be expressed in plants, they have a cell wall (hard to extract), and the glycosylation process is different.
What makes mammalian cell culture systems challenging for protein expression
Mammalian cells have complex nutritional requirements, grow slowly, and are easy to contaminate.
Why are mammalian cell culture systems still the best choice for protein expression?
Mammalian cells are the best choice for producing proteins that are destined for use in humans.
What is an animal bioreactor production system used for in protein expression?
for monoclonal antibody production; inject mice with an antigen and purifying the secreted antibody.
What is an antibody?
An antibody is a protein produced in response to antigens of invading viruses or bacteria, which combines with and neutralizes the antigen.
What role do antibodies play in the immune system?
Antibodies protect the organism by neutralizing antigens, helping resist infectious diseases as part of the immune response.
What are baculoviruses used for in protein expression?
Baculoviruses are used as vehicles to insert mammalian DNA into insect cells, leading to the production of desired proteins.
How do post-translational modifications of proteins differ in insect systems compared to mammals?
The post-translational modifications of proteins can be slightly different in insects than in mammals.
When is the insect system typically used in protein expression?
The insect system is currently used when small quantities of proteins are needed for research.
What is the first step in protein purification during downstream processing?
Preparing an extract for purification, which involves harvesting entire cells if the protein is intracellular (requiring cell lysis) or collecting culture medium if the protein is extracellular.
How is cell lysis achieved when preparing intracellular proteins for purification?
Methods include freeze/thaw cycles, detergents, mechanical methods, and the addition of organic alcohols and salts to disrupt the cell wall and release the protein.
How are extracellular proteins prepared for purification?
he culture medium is easily collected because the protein is excreted into the medium and can be recovered with a pipette.
What is the second step in downstream processing protein purification?
Stabilizing proteins in solution by maintaining low temperature and proper pH, adding protease inhibitors and antimicrobials, and using additives to prevent foaming and shearing.
Why are protease inhibitors and antimicrobials added during the stabilization of proteins in solution?
To prevent protein digestion during purification.
What is the third step in downstream processing protein purification?
Separating components in the extract by exploiting similarities between proteins to separate them from other macromolecules BASICALLY FILTERING
How does protein precipitation work as a separation method in downstream protein purification?
Hydrophilic amino acids on protein surfaces attract water, and salts like ammonium sulfate or solvents like ethanol are added to precipitate proteins by removing water.
What role do salts and solvents play in protein precipitation?
Salts such as ammonium sulfate and solvents like ethanol or isopropanol cause proteins to precipitate by removing water from between the protein molecules.
What size-based filtration methods are used for protein separation in downstream processing?
Centrifugation and membrane filtration (diafiltration), with methods such as microfiltration and ultrafiltration to remove unwanted debris and separate large proteins from small ones.
What is the difference between microfiltration and ultrafiltration in protein separation?
Microfiltration removes precipitates and bacteria, while ultrafiltration separates large proteins from small proteins.
What is a potential problem with membrane filtration in protein purification?
Membrane filtration is prone to clogging, especially during ultrafiltration.
What is the purpose of dialysis in protein purification?
Method of protein separation that removes smaller salts, solvents, and additives by allowing smaller molecules to pass through a semi-permeable membrane while retaining larger molecules like proteins.
How does dialysis work in protein purification?
It relies on the chemical concept of equilibrium, where dissolved substances migrate from areas of high concentration to lower concentration through a semi-permeable membrane.
When salts are removed in dialysis, what are they replaced with?
Buffering agents to stabilize proteins for the rest of the process.
What is chromatography used for in downstream protein purification?
It sorts proteins based on their size or how they bind to other substances as they pass through resin beads in a glass column.
How does size exclusion chromatography separate proteins?
It uses porous gel beads as a filter system where large proteins work around the beads and small proteins enter the beads, moving slower.
How does ion exchange chromatography work in downstream protein purification?
It relies on electrostatic charge to bind proteins to resin beads, where contaminants pass through and proteins are later released by increasing the salt concentration.
What are the differences between anion exchange and cation exchange resins?
Anion exchange resin is positively charged and binds negatively charged proteins, while cation exchange resin is negatively charged and binds positively charged proteins.
How does affinity chromatography separate proteins?
It relies on the specific and reversible binding of proteins to ligands, with buffer solutions used to wash out unbound molecules and release retained proteins.
What is the advantage of affinity chromatography in protein purification?
It shortens the purification process and highly selective
What is hydrophobic interaction chromatography?
It separates proteins based on their repulsion of water, where hydrophobic amino acids in the protein are attracted to hydrophobic molecules coated on the column beads.
What is 2-dimensional gel electrophoresis used for in protein purification?
It separates proteins based on their isoelectric point and molecular weight, allowing for the separation of similar proteins from each other.
What is the isoelectric point in 2-dimensional gel electrophoresis?
separation of proteins based on their unique electronic signature.
What is SDS-PAGE used for in protein purification?
It separates proteins based on size and mass by adding SDS detergent, which evenly distributes sulfate charges along the protein, making separation dependent on size.
How are proteins visualized in SDS-PAGE? (polyacrylamide gel electrophoresis)
After separation, proteins are stained with Coomassie stain to make them visible.
What is the purpose of Western blotting in verifying protein purification?
Western blotting is used to detect specific proteins by transferring them from SDS-PAGE to a membrane.
How are proteins detected in Western blotting?
Primary antibodies bind to the protein of interest, and a secondary antibody coupled to a reporter enzyme is added. A substrate is then used to visualize the bound antibodies.
