1.27.2010 lecture
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Greg 1. DNA or RNA probes can be used to detect specific sequences of interest in cells through techniques like Southern blots, Northern blots, and in situ hybridization. 2. PCR and RT-PCR can be used to amplify nucleic acid sequences, allowing detection of low concentrations. Reverse transcriptase is used to generate cDNA from mRNA. 3. Techniques like overexpression and tagging of proteins allow studying the function and localization of proteins in cells. Fluorescent tags like GFP enable visualization of proteins in living cells.
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The document discusses DNA cloning techniques. It describes how DNA cloning allows for the mass amplification and stable propagation of specific DNA sequences. The key steps involve using restriction enzymes to cut DNA fragments and plasmids, ligating the DNA fragment into the plasmid, transforming host cells with the recombinant plasmid, and selecting for transformed cells. Plasmids are commonly used as cloning vectors due to their small size, circular structure, and ability to replicate independently of the host cell genome.
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Genome editing tools form the basis for personalized medicine, especially for therapies requiring change in genome. Currently there are four contenders to this – Meganucleases, ZNF Nucleases, TALENs and CRISPRs. Although, the technologies are many, there are very few commercial providers of this technology. This is attributed to the fact that select few possess the intellectual property rights of turning these technologies to valid form of therapy; for example, ZFN patent with Sangamo BioSciences and TALENs with Cellectis, Transposagen and Life Technologies.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
Nucleic acid and Molecular markers
The document discusses nucleic acids and their components. Specifically:
- Nucleic acids are essential biomolecules composed of nucleotides, which contain a nitrogenous base, pentose sugar, and phosphate group.
- DNA and RNA are the two main types of nucleic acids. They are made up of nucleotides and have characteristic bases and pentoses.
- RNA has several types including mRNA, rRNA, and tRNA that play important roles in protein synthesis and ribosome structure.
Restriction mapping
DNA fingerprinting involves detecting variations in DNA sequences between individuals to generate unique profiles. There are two main categories of techniques: hybridization-based like Restriction Fragment Length Polymorphism (RFLP) and PCR-based like Randomly Amplified Polymorphic DNA (RAPD). RFLP was the first technique developed and involves digesting DNA with restriction enzymes and comparing fragment lengths on gels. PCR-based techniques do not require blotting/hybridization and can use smaller DNA quantities. DNA fingerprinting has applications in forensics, genetics, and plant/animal breeding.
Techniques Used in Recombinant DNA Technology
This assignment cum Infographic presents you with vital knowledge about recombinant dna technology, its scope, its goals & objectives & the techniques uesd in it. I have referred & combined essential information from from various journals, infographics,essays, published papers of eminent researchers & scientists in this
Lab Differential Expression Differential gene expression provides th.pdf
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Lab Differential Expression Differential gene expression provides .pdf
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Biotechnology Ap
1. The document discusses genetic engineering and biotechnology, including techniques like recombinant DNA, gene cloning, PCR, and cloning organisms.
2. Key terms defined include genetic engineering, biotechnology, recombinant DNA, gene cloning, restriction enzymes, and plasmids.
3. Recombinant DNA is made by cutting DNA with restriction enzymes and ligating pieces into plasmids, which are then inserted into bacteria.
DNA cloning
DNA cloning techniques allow for the reproduction of DNA fragments. There are two main approaches: cell-based cloning using living cells and in vitro cloning using PCR. The cloning process involves cutting out the gene of interest and a host plasmid with the same restriction enzyme, ligating the gene into the plasmid, transforming bacteria with the new plasmid, and selecting for transformed colonies. Common cloning vectors include plasmids, bacteriophages, cosmids, YACs, and retroviral vectors which allow incorporation of foreign DNA into host genomes. PCR cloning strategies like TA cloning take advantage of Taq polymerase adding extra adenines to PCR products, allowing ligation into vectors with thymidine overhangs.
Similar to 1.27.2010 lecture (20)
DNA FINGERPRINTING TECHNIQUE FOR IDENTIFICATION OF DRUGS OF NATURAL ORIGIN AN...
DNA FINGERPRINTING TECHNIQUE FOR IDENTIFICATION OF DRUGS OF NATURAL ORIGIN AN...
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
Lab Differential Expression Differential gene expression provides th.pdf
Lab Differential Expression Differential gene expression provides th.pdf
Lab Differential Expression Differential gene expression provides .pdf
Lab Differential Expression Differential gene expression provides .pdf
More from Greg
Test 1 article
The document summarizes a study that used live cell imaging to visualize hepatitis C virus (HCV) entry into human liver cells. Key findings include:
1) HCV was labeled with fluorescent membrane dyes and found to move along actin stress fibers in an actin-dependent manner after internalization, suggesting clathrin-mediated endocytosis.
2) Inhibition of clathrin machinery or related host factors blocked HCV entry. Immunofluorescence showed HCV co-localizing with clathrin and early endosomes.
3) While tight junction proteins were thought to mediate HCV entry, live imaging found internalization occurring outside of tight junctions, indicating their role may be overstated.
4
Test article 2
The document discusses mitochondrial dysfunction in insulin resistant mice (DKO mice) as a model for type 2 diabetes. Electron microscopy showed DKO mice had larger, fewer mitochondria than controls. Foxo1, a transcription factor regulated by insulin, was dysregulated in DKO mice, affecting genes controlling mitochondrial function. Immunoblotting showed altered levels of fission/fusion proteins in DKO mice mitochondria. Respiration and ATP production were lower in DKO mice mitochondria, as was electron transport chain activity. Ppargc1a, a regulator of mitochondrial biogenesis, was highly expressed but inhibited by acetylation in DKO mice. Restoring Ppargc1a function or deleting Foxo1 improved mitochondrial morphology and
Review session for test 2
This document provides an overview of phospholipid formation and the Krebs cycle. It discusses how enzymes transfer fatty acids from fatty acyl CoA to glycerol-3-phosphate to form diacylglycerol. Diacylglycerol is then used with choline transferase to generate phosphatidylcholine, a major phospholipid. It also lists oxaloacetate as the starting point of the Krebs cycle and notes the steps that yield NADH and GTP, as well as the regulatory steps involving changes in Gibbs free energy.
3.1.2010
The document summarizes key aspects of protein transport within cells, including:
1) Proteins entering the endoplasmic reticulum (ER) have an N-terminal signal sequence that is recognized by signal recognition particle (SRP) to initiate translocation. Chaperone proteins such as BiP assist with proper folding within the ER lumen.
2) Transmembrane proteins maintain their orientation throughout transport, with alpha helices embedding in membranes. Multiple transmembrane proteins have multiple start and stop sequences recognized by SRP.
3) Vesicle transport between organelles is mediated by SNARE proteins on transport vesicles (v-SNAREs) and target membranes (t-SNAREs) that facilitate docking and fusion
2.26.2010
Peroxisomes are organelles that oxidize fatty acids, producing hydrogen peroxide (H2O2), which they then decompose to water and oxygen using the enzyme catalase. They can also utilize organic compounds as a source of hydrogen. Peroxisomes replicate through division or can form de novo from the endoplasmic reticulum. Proteins destined for peroxisomes contain a peroxisome targeting signal (PTS1) that directs them to the organelle.
2.24.2010
The document summarizes key processes that occur in the endomembrane system. It describes how the rough endoplasmic reticulum synthesizes proteins and lipids. It then discusses post-translational modifications that occur in the Golgi apparatus, including glycosylation. From the Golgi, vesicles traffic proteins to other organelles or secrete them. Lysosomes contain enzymes to break down molecules. Autophagy and phagocytosis allow degradation of old organelles or foreign particles respectively. Peroxisomes contain enzymes to break down toxic metabolites.
2.22.2010
Actin filaments interact with myosin proteins to enable cell movement. Specifically, myosin I interacts with actin filaments and transports vesicles along actin filaments, moving toward the plus end. When the globular heads of myosin bind to actin filaments, they cause the filaments to bend inward toward the minus end, shortening the sarcomere. Myosin II is involved in cell division. There are several steps to cell movement, including formation of a leading edge and protrusion of lamellipodia and filopodia, which adhere to actin filaments via myosin I, and severing and contraction of actin filaments at the cell rear mediated by myosin II.
2.22.2010 organelles
Cells create organelles that concentrate proteins and enzymes in specific areas. The endomembrane system includes the endoplasmic reticulum, Golgi apparatus, and transport vesicles. The endoplasmic reticulum buds vesicles that are sent to the Golgi apparatus, then to the plasma membrane. Isolated organelles include the nucleus and mitochondria.
2.19.2010
1) Microtubules grow by the addition of tubulin dimers to the plus end, with polymerization favored at this end. The plus end carries a GTP cap that stabilizes the microtubule.
2) Microtubules undergo treadmilling as the GTP cap moves down the length of the microtubule until reaching the minus end, where GTP is hydrolyzed.
3) Dynamic instability occurs when a microtubule switches between growth and shrinkage, providing a source of tubulin for other microtubules. Growing microtubules are favored when GTP-tubulin concentrations are high.
2.17.2010
1) The cytoskeleton is made up of microtubules, microfilaments, and intermediate filaments that regulate cell structure, motility, and transport of organelles.
2) Microtubules are involved in cell division and shape cellular structures like cilia. Microfilaments help with cell motility and division. Intermediate filaments provide structural support and strength to cells.
3) Mutations in proteins that make up the cytoskeleton can cause diseases like epidermolysis bullosa and Lou Gehrig's disease that impact skin, muscle, and nerve cells.
2.12.2010
The document summarizes key aspects of cellular respiration, including:
1) Glycolysis can be reversed through gluconeogenesis to generate glucose from other sources.
2) Pyruvate enters the mitochondrial matrix and is converted to acetyl-CoA, which fuels the Krebs cycle to generate NADH, FADH2, and GTP while producing CO2.
3) The electron transport chain uses the energy from NADH and FADH2 to pump protons out of the mitochondrial matrix, creating an electrochemical gradient that drives ATP synthase to generate ATP from ADP and phosphate.
2.10.2010
1. Glycolysis converts glucose to pyruvate through a series of 10 steps, generating 2 ATP and 2 NADH per glucose molecule.
2. Key regulated steps include conversion of glucose to glucose-6-phosphate which traps glucose in the cell, and conversion of fructose-6-phosphate to fructose-1,6-bisphosphate which controls glycolysis.
3. Pyruvate generated by glycolysis can then undergo fermentation, producing lactate or ethanol to regenerate NAD+ and allow glycolysis to continue under anaerobic conditions.
2.8.2010
1) Active transport uses energy, usually from ATP hydrolysis, to move molecules against their concentration or electrochemical gradients. This includes direct transporters that directly hydrolyze ATP and indirect transporters where one molecule moves down its gradient to drive another up its gradient.
2) Passive transport utilizes concentration and electrochemical gradients to move molecules down their gradients and does not require energy. Facilitated diffusion uses carrier proteins to increase the rate of diffusion for molecules.
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3.19.2010
Small G proteins bind either GDP or GTP and function as molecular switches. When bound to GTP, they interact with and activate target proteins. Heterotrimeric G proteins are composed of alpha, beta, and gamma subunits and bind to G protein-coupled receptors in the cell membrane. When a ligand binds the receptor, it activates the G protein, causing the alpha subunit to exchange GDP for GTP. The alpha subunit then activates downstream effector molecules like adenylyl cyclase. Adenylyl cyclase converts ATP to cyclic AMP (cAMP), which activates protein kinase A and triggers phosphorylation of target proteins to elicit cellular responses.
3.24.2010
The document summarizes key concepts about the cell cycle, including:
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2) Experiments fused cells at different phases and observed activation, identifying maturation promoting factor (MPF) as important for mitosis.
3) Further work identified MPF as a cyclin-Cdk complex, with cyclin levels and Cdk activating phosphorylations regulating progression through the cell cycle.
4) The cyclin B-Cdk1 complex promotes mitosis, driving events like nuclear envelope breakdown, before cyclin degradation inactivates Cdk1
3.22.2010
The document provides an overview of calcium regulation and receptor tyrosine kinase (RTK) signaling pathways. It describes:
1) Calcium is maintained at low levels (~10^-7M) in the cytoplasm and stored at higher levels (~10^-3M) in the endoplasmic reticulum. Calcium is released through IP3 and ryanodine receptors on organelles and voltage or ligand gated channels on the plasma membrane. Calcium is transported back into storage by ATPase pumps.
2) RTKs dimerize and autophosphorylate when bound by extracellular ligands such as growth factors. This recruits adapter proteins like Grb2 and SOS, activating the Ras/MAPK pathway through
3.17.2010
The document summarizes key aspects of signal transduction pathways involving smooth muscle relaxation. It describes how acetylcholine binds to receptors on endothelial cells, activating a G protein which signals the production of nitric oxide (NO). NO then diffuses into the smooth muscle cell where it binds receptors and activates guanylate cyclase, increasing cGMP levels. cGMP activates protein kinase G, which phosphorylates calcium channels and causes them to close. With reduced calcium levels, the smooth muscle relaxes. The document also provides an overview of different receptor types, molecular switches in pathways, and differences between heterotrimeric and small G proteins.
3.15.2010
The document discusses different types of cell signaling including endocrine, paracrine, contact-dependent, and intracellular signaling. It describes various signaling molecules such as hormones, growth factors, cytokines, neurotransmitters, and gases that act as signals between cells. These molecules bind to receptors on target cells and induce downstream signaling cascades and cellular responses such as changes in gene transcription or cytoskeleton that affect processes like cell survival, proliferation, and death.
3.5.2010
1. Vesicles bud off from donor membranes with v-SNAREs and are carried along microtubules by motor proteins to target membranes containing t-SNAREs.
2. Vesicle and target membrane tethering proteins bring the two close together to allow v- and t-SNAREs to associate and fuse the vesicle and target membrane.
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Provenge is a cancer immunotherapy that involves extracting dendritic cells from a patient, shipping them to a company where they are treated with a prostate cancer antigen and GM-CSF growth factor, and then re-injecting the activated dendritic cells into the patient in 3 injections. The document also defines and compares the intrinsic and extrinsic apoptosis pathways, describes the Ras signaling pathway and its role in cell survival and proliferation, and outlines the stages of the cell cycle including checkpoints at G1/S and G2/M.
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1.27.2010 lecture
- 2. You must know nucleic acid or gene, you must know the sequence to make a single stranded probe.
- 3. Can do hybridization procedures with the single stranded DNA.
- 4. % formamide and temperature. 50% 37 degrees C may result in high pairing.
- 5. 50% 35 degress C may result in imperfect matching in double helix.
- 6. Can then pull out region of interst.
- 7. Can do same thing with specific sequences of RNA
- 8. Riboprobes - Produced by in vitro transcription.
- 9. Take sequence of DNA and insert into plasmid behind a promoter construct that is regulated by viral RNA polymerases.
- 10. Bacteria incorporate the plasmid and the viral bacteria will continue to transcribe the DNA into RNA.
- 11. Label the nucleotides so when the RNA is made, it’s labeled.
- 12. Dedoxygenin (marker) that is converted into a precipitate by enzymes so you can visualize in the cell
- 13. Radioactive, fluorescent, and enzymatic markers.
- 14. In this transcription process, you make sense and antisense molecules.
- 15. How are the DNA or RNA probes used?
- 16. To detect the DNA or RNA of interest in the cell.
- 17. Northern blots are used to identify RNA – don’t localize or tell where in the cell a protein is.
- 18. Same procedure as southern blot, except using different buffers, and lets you see RNA
- 19. Southern blots are used to identify DNA - don’t localize or tell where in the cell a protein is.
- 20. Take DNA in cell, denature, expose to radioactive probes that attach to DNA that has been transferred onto filter. Probes only target the sequences you are interested in. Rinse excess radioactivity/fluorescence.
- 22. Probe is localized in the cell to a specific site and then you can view the genes or sequences while they are still in the living cell/chromosome.
- 23. FISH
- 24. (fluorescence in situ hybridization) is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes are. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
- 25. Genes localized by specific probes
- 26. Can see in a chromosome
- 27. Can label RNA in a cell
- 28. How do you amplify samples if your proteins are scarce?
- 29. PCR
- 30. Usually only 1 sample
- 31. RT-PCR
- 32. More than 1 sample
- 33. Identify the presence of nucleic acids in low concentrations.
- 34. Reverse transcriptase polymerase chain reaction
- 35. Start with message from sample.
- 36. Collect all mRNA
- 37. Hybridize with a poly-t-primer at end of sample that will reverse transcribe cDNA
- 38. Uses single stranded mRNA as template
- 39. Degrade RNA strand with tail and left with cDNA
- 40. Use polymerase to amplify cDNA sequence
- 41. Now you have a double cDNA copy.
- 42. Amplify
- 43. Separate cDNA using high temps (95) and cool down (50) to add primers, which gives the DNA polymerase something to hang on to.
- 44. DNA polymerase is heat stable, but will only work at about 70 degrees C.
- 45. Add nucleotides to sample so the DNA polymerase can use to make cDNA strand.
- 46. Tac polymerase – needs Mg2+, nucleotides, single stranded template, double stranded region of DNA.
- 47. Amplification of about 10^9 times.
- 48. Must know what sequences you’re looking for…
- 49. Micro-arrays
- 50. Whether genes are upregulated or down regulated in certain conditions.
- 51. Takes advantages of chips that are generated where you have multiple single stranded copies of genes with themes (growth factors, cytokines etc)
- 52. Take samples and isolate mRNA then convert to cDNA and label with fluorescent probe. Hybridize to microarray. Where are the green/red/yellow spots? The key for the chips tells which single stranded DNA that is or which gene is that that has been bound to a cDNA construct from sample XYZ. Targets differences between two samples or in certain conditions.
- 53. Techniques used to alter protein expression
- 54. What is the role of a protein in cell?
- 55. Overproduce a protein to determine its function
- 56. Use DNA plasmid vectors
- 58. Cut plasmid
- 59. Insert particular DNA sequence behind promoter
- 61. Plasmid can be expressed and make protein of interest
- 62. Protein function can be dependent on concentration, so be careful.
- 63. Will misinterpret if there is a negative outcome
- 64. In order to determine if protein has been expressed, cut, antibodies on western blot to determine level of expression, correlate back to determine changes in cell
- 65. Or … Identify protein of interest specifically by tagging
- 66. Insert gene of interest into a construct that has a particular tag, such as histidine tags with Ni -HA tags – Histidine w/ nickel
- 67. M2 tags – protein structure is recognized by flag antibody
- 68. GST tag – glutiphinyl s transferase tag, can also be identified by antibodies for GST
- 69. Look for tag itself on western blot
- 70. Antibodies – tag so you don’t need to generate an expesivie antibody for protein
- 71. Purifies protein rapidly, tags separate on column.
- 72. Make sure tag isn’t doing anything to cell function
- 73. How to use proteins, that are themselves fluorescent, to identify proteins of interests
- 74. GFP is a tag that has a construct that has an inducible promoter and has several sites for restriction endonucleases to insert DNA of interest. The construct won’t be expressed until you add the sugar that activates it. ITPG sugar activates lac promoter and the lac promoter begins to turn on transcription and translation of the protein plus GFP.
- 75. You can use living cells with GFP and fluorescent microscopy.
- 76. We can insert a gene of interest and we have a gene that confers drug resistance to those cells that are transfected with the plasmid. Not all cells stably transfect into genome. Those that have, will have ampicilin resistance. After a while, only those that have stably incorporated live. All others die off.
- 77. Maybe you wanna eliminate protein function.
- 78. Replacement – difficult in mammalian cells
- 79. Knockout to remove active gene – difficult in mammalian, so you use embryonic stem cells injected into target gene replaced by mutation to generate progeny in a pregnant mouse that now expresses mutant/altered gene. Some have the mutation. Mate mice with normal mice so in F2, look for males and females with copies of mutated genes. Can be in somatic cells. Transgenic mice are looked at for the function of that gene.
- 80. Add a competing gene (gene addition)
- 81. RNAi
- 83. Double stranded RNA has been known to be considered viral.
- 84. Double stranded RNA will be targeted by dicer, which cleaves the ends of the double stranded RNA making an siRNA (small interference RNA). The presence of siRNA is an indicator of these mechanisms of infection.
- 85. RISC proteins attach to siRNAs and degrade 1 strand of siRNA leaving a single strand which can:
- 86. Bind to message that has been generated which inhibits translation
- 87. Enter nucleus and bind complementary DNA to inhibit transcription.