Exam 1 Flashcards
Ethics
“Conforming to accepted and professional standards of conduct”
Taking an NIH _____ _____ course is a federal requirement for graduate school!
Research Ethics
Violations of academic integrity
Copying, sharing, attempting to get an unfair advantage over others, stealing, lying, plagiarism, lack of attribution, tampering, non witness, false witness, falsifying, fudging, enhancing, etc.
Deliberate plagiarism
Rewriting from books or articles, copying and pasting from web pages and online sources to create patchwork writing, buying, downloading, or borrowing a paper.
Accidental Plagiarism
Not knowing when and how to cite, not knowing how to paraphrase or summarize, not knowing what common knowledge is, recycling an old paper
How do scientists mess up?
Honest errors of measuring, recording, interpreting, lack of review, overinterpretation/lack objectivity, pressure to produce, lack of responsibility, plagiarism, purposeful errors of fabrication, omission, falsification
How do we stay on the right track?
Double check and triple check data, objectivity and standards, mentoring, review by senior author, affirming authorship and contributions, checking of submitted verbs and figures
Consequences of violations
Loss of credibility, retract public work, loss of degree or job, barred from funding, jail and fines, damage field
Immunology
Study of the structures of the immune system
Immune system cells
Lymphocytes, killer T cells, helper T cells
Immune system organs
Thymus, lymph nodes, peyer’s patches (gut)
Immune system physical barriers
Skin, mucus, and keratin
Innate immune response
No prior contact required, includes inflammation
Adaptive immune response
Reacts to antigens, remembers an invader, highly specific to antigen, takes time to acquire
B cells
Secrete antibodies, mount the humoral immune response
T cells
Attack cells infected with bacteria or viruses. Cell-mediated immune response
Antigens
Not usually part of the host, large polysaccharides or proteins on the sura=faces of viruses or foreign cells.
Epitope
Specific region on antigen to which the antibody binds. Linear or conformational.
Antibodies
Secreted by B cells, recognize epitopes on antigens, binds to antigens to make them recognizable, leads to neutralization and/or destruction of the antigen
Antibody molecule structure
Y-shaped, with two antigen-binding sites, specific to the antigenic determinants that elicited its secretion. Also called immunoglobins
Effects of antibody binding
Inactivation of antigens by neutralization, agglutination of microbes, and precipitation of dissolved antigens (all enhance phagocytosis), or activation of complement system (leads to cell lysis)
Antibody applications: Purification
Mix cell “lysate or extract” with antibodies, use antibodies to purify molecules
Immunoprecipitation
Add specific anti-A antibodies to mixture of molecules, collect aggregate of A molecules and anti-A antibodies by centrifugation
SDS-Page
(Sodium-dodecyl sulfate – polyacrylamide gel electrophoresis), Proteins forced through gel by electric current. Move towards the positive end as proteins are negatively charged.
Western Blotting
Antigen A separated from other molecules by electrophoresis, incubation with labeled antibodies that bind to antigen A allows position of antigen to be determined. Sensitivity increased by using multiple layers of antibodies.
How are antibodies made?
Injecting an animal with antigen A over several weeks stimulates specific B cells large amounts of this. Many different B cells will be stimulated.
Polyclonal antibodies
When an antigen such as a protein is injected into an animal, a mixture of antibodies is produced an isolated. Each antibody in the mixture recognizes a different, specific epitope within the protein.
Monoclonal antibodies
Identical antibodies to a specific epitope of protein. Produced by a clone originating from one cell. More specific, stronger data, more expensive.
ELISA
Enzyme Linked Immunosorbent Assay. Diagnostic tool for infectious diseases, track pathogens in animals, plants, etc.
ELISA applications
Detects GMOS, allergens, pregnancy, drugs
Primary antibodies
Bind directly to antigen in question.
Secondary antibodies
Bind to primary antibody, conjugated to a fluorochrome or enzyme for colorimetric or chemiluminescent detection. Must be made in a different animal from primary and be immunoreactive to the antibody it will bind to.
A second set of antibodies created to target the Fc fragment (constant region) of the primary antibody
Purpose of secondary antibodies
Provide an additional step for signal amplification, increasing overall sensitivity of assay
ELISA Reagents and materials
Solid support, blocking reagent, primary and secondary antibodies.
ELISA solid support
Microplate strips made of polystyrene that bind proteins via hydrophobic interactions
ELISA Blocking reagent
Used after binding of antigen. Prevents binding of antibodies to plate. Eliminates or reduced false positives, prevents non-specific binding.
ELISA Primary antibody
Rabbit polyclonal anti-gamma globulin
ELISA Secondary antibody
Goat anti-rabbit IgG. Coupled to horseradish peroxidase (HRP)
Horseradish Peroxidase
Catalyzes the oxidation of the chromogenic substrate 3,3’, 5.5’-tetramethylbenzene (TMB). Also requires presence of hydrogen peroxide.
ELISA False positive causes
Pipetting error, contamination, recent vaccination
ELISA False negative causes
Immunosuppression, testing too soon, denatured antigen or antibody
Immune response
The ability of an organism to both defend and recognize itself
Humoral response
- Lymphocytes originate from stem cells in bone marrow
- Lymphocyte stem cell matures into B-cell in the bone marrow
- When B-cells encounter antigens, they become B-plasma cells that secrete antibodies and confer immunity
Cell-mediated response
- Lymphocyte stem cell matures into a mature T-cell in the thymus and enters circulation
- T-cells have specific receptors that bind to antigens
- Attack infected host cells via binding followed by cell lysis
Three antibody regions
Constant, variable, and joining regions
constant region
conserved region
Variable region
Region confers specificity to the binding interaction with the antigen
Joining regions
Regions that connect the constant and variable regions
Enzyme substrate
Tetramethylbenzidine (TMB)
Enzyme catalyst
Hydrogen peroxide
What does HRP do in ELISA?
Cleaves TMB in presence of hydrogen peroxidase to create blue byproduct, allows for visual confirmation of results
Triplicate samples
Another form of control, same results should be in all three wells. Checks for accuracy.
Location of DNA in plant cells
Nucleus, mitochondria, and chloroplast
PCR mimics in vitro replication:
- DNA unwinds
- Exposes single stranded areas to be copied (ssDNA)
- Primer used to initiate replication
- DNA polymerase adds nucleotide base pairs (A, T, C, G) to make new complementary strand by using ssDNA as template
PCR vs. DNA replication
Uses the same ingredients and methods of cellular DNA replications but uses temperature changes to complete the process
PCR steps
Denaturation, Annealing, and Extension
Denaturation
Heat melts double stranded DNA into ssDNA by breaking the H-bonds holding the base pairs together
Annealing
Temperature’s lowered, primer sticks to DNA
Primer design for PCR
Forward primer, amplified region, and reverse primer
Extension
DNA polymerase adds nucleotides (A, T, C, G) to the attached primer to form a new complementary strand, using the ssDNA as a template. Extends in a 5’ –> 3’ direction
Taq (Thermus aquaticus) polymerase
Polymerase isolated from a bacterium living in hot springs. Ideal because it’s a thermostable enzyme.
Magnesium chloride
Taq polymerase cofactor
Reaction Buffer
Maintains pH, contains salt
End-point PCR
DNA must first be separated by gel electrophoresis. DNA staining and detection are done after PCR.
Real-time PCR
Product is measured during the PCR. No electrophoresis required. Can be quantitative when compared to a standard curve.
Uses a fluorescent dye that binds to double stranded DNA or a fluorescent probe that’s specific to the target sequence
Fluorescent detection
Requires no gel electrophoresis, higher throughput, more sensitive, faster than PCR
GMOs
“An organism in which the genetic material has been altered in a way that doesn’t occur naturally by mating or natural recombination”
How are GMOs made?
- Identify potentially beneficial gene
- Isolate and clone the gene
- Engineer the gene: Add promoter and terminator, add selectable marker
Promotor we used
CaMV 35S (from Cauliflower mosaic virus), functions in every plant cell
Terminator we used
NOS (Nopaline synthase). From soil bacterium Agrobacterium tumefaciens, recognized in most plants.
Selectable marker we used
GFP or antibiotic resistance. Used to determine which cells contain the engineered gene.
Current transgenic plants
Corn, alfalfa, soybeans, cotton, sugar beets, papaya, squash
Pros of GMOs
Reduce the use of pesticides and herbicides, reduce stress to the land, improve nutritional value of food, allow previously unfarmable land to be used.
Cons of GMOs
Time consuming and inefficient, expensive, superweeds and superbugs, allergic reactions and antibiotic resistance, not enough research, lack of government requirements for food labeling
Instagene matrix
Chelates divalent ions (Mg 2+) necessary for DNA degrading enzymes (Ex: DNAses)
Target DNA to detect GMO
CaMV promoter, Nos promoter
Target DNA to check DNA extraction
Photosystem II gene
GMO false positive reasons
Contamination
GMO false negative reasons
Failed DNA extraction, incorrect primers, failed PCR
Electrophoresis
Way to separate DNA molecules of different sizes
How does Agarose work?
Provides a solid matrix with pores. Buffer solution with DNA goes through the pores, and DNA is separated as it tries to pass through.
Agarose
Gelling component, polysaccharide polymer made from seaweed extract.
Ogston Sieving
Regards the DNA molecule like a tangle of thread. Smaller molecules fit into more pores, travel faster.
Reptation
Regards the long DNA molecule as a snake. The longer the DNA strand, the longer it takes because its route is more complicated.
Typical agarose gel density
1%. The higher the gel percentage, the longer the run time necessary to separate fragments.
DNA loading buffer
Added to sample to give it color and density. Contains colored dye, thickening agent, and EDTA.
Colored dye
Tracks the progression of the samples down the gel. (Bromophenol Blue)
Thickening agent
Samples need to be denser than water so they’ll sit in the wells. Sucrose and/or glycerol are added to provide weight.
EDTA
Binds Mg+ and stops all subsequent enzyme reactions (nucleases)
High voltage results
Faster, but too high and gel will melt.
Low voltage results
Looks better (tight bands), but can take awhile.
Ethidium Bromide
Binds to DNA and a UV light illuminates the dye. Carcinogenic.
DNA ladders indicate _____
fragment size
Factors affecting DNA migration
Size of DNA, DNA conformation (supercoiled vs. linear), ionic strength of running buffer, voltage applied to gel, concentration of gel, electroendosmosis
Electroendosmosis
Charged molecules in agarose, impede migration of DNA. USe of ultrapure agarose with low sulfate and carboxylate content can reduce this.
Other applications of DNA gel electrophoresis
Verify DNA extracts (ensure not contaminated with RNA)
Verify PCR products (Check for mis-amplification)
Sanger sequencing, genetic fingerprinting
DNA Barcoding
Using DNA sequences to determine species. Species occur in same time and space
Morphological species
Based on physical characteristics. Doesn’t always work (Mimicry, traits change with age, sexual dimorphism), not efficient because all species have to be stored for comparison
Biological species
Based on who can mate with each other. Doesn’t always work because hybrids, extinct species can’t mate.
Phylogenetic species
Based on evolutionary history.
DNA polymorphism
Changes in genetic code
DNA barcoding animal primer
Cytochrome c oxidase subunit 1. DNA source is mitochondrion
___ interspecies variation, ___ intraspecies variation
high, low
What primer is used to sequence in DNA barcoding?
M13
BLAST (Basic local alignment search tool)
One of the most commonly used bioinformatics software. Finds small sub-sequences of the query in the subject sequence.
Uses word to match with the database of subject and then uses heuristics to verify and extend match. Compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches.
Heuristic
A rule/method that helps you solve problems faster than you would if you did all the computing
Bit score
Score based on alignment, length of sequence, and length of database. The bigger the number, the better, but a high score doesn’t always mean the query’s unique. Score of sequence similarity, not a score of sequence homology.
E-value
Expected amount of random sequences that have equivalent sequence alignment. Calculated using the max bit score and the length of the query and database. Tells you relative strength of alignment. The lower the better.
____ sequences have higher e-values because probability of finding that sequence is higher.
shorter
Risk Group 1
Agents are not associated with disease in healthy adult humans
Risk Group 2
Agents are associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available
Risk Group 3
Agents are associated with serious or lethal human disease for which preventative or therapeutic interventions may be available
Risk Group 4
Agents are likely to cause serious or lethal human disease for which preventative or therapeutic interventions are usually available
Containment
Used in describing safe methods for managing infectious materials in the laboratory environment.
Primary containment
The protection of personnel and the immediate laboratory environment from exposure (Good microbiological technique, use of PPE and safety equipment)
Secondary Containment
The protection of the environment external to the laboratory from exposure (facility design, operational practices)
Biosafety level 1
Wash hands, protective clot, prohibit eating, drinking, and smoking. Policies for safe sharps handling, decontaminate work surfaces daily
Biosafety Level 2 standard practices
Same as BSL-1 with emphasis of face/eye protection, gloves, mechanical pipetting, attention to sharps. Has hooded cabinets
Biosafety Level 3
Secondary barriers plus anteroom, negative air flow, and autoclave within the lab
Biosafety level 4
Positive pressure personnel suit with segregated air supply, UV room, multiple showers.
Orange bags
Biohazard. Sterilized in autoclave, no sharps, Labeled and treated, then disposed of in regular trash.
Red bags
Infectious waste. Secured in secure area with impervious flooring. Later labeled and incinerated in special facility.
