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What is the goal/application of PCR?
the goal of PCR is to amplify a specific gene
PCR= polymerase chain reaction
What can we use PCR for?
DNA fingerprinting
Classification of organisms
Genotyping
Prenatal diagnosis
Mutation screening
Drug discovery
Bioinformatics
Genomic cloning
Human Genome Project
Site-directed mutagenesis
Gene expression studies
PCR
polymerase chain reaction
What are the three steps in a PCR application, and the components required to set up a PCR?
components: DNA sample, primers, nucleotides, enzyme (polymerase), buffer, PCR tube, mix buffer
process: denaturing, annealing, extension
what happens during denaturing?
2 strands of DNA separate
what happens during annealing?
primers bind to template
what happens at extension?
synthesize new strand
at what temperature does denaturation occur?
95 C
at what temperature does annealing occur?
55 C
at what temperature does extension occur?
72 C
Understand how you would set up appropriate positive and negative controls in a PCR.
positive control- a sample that has been validated over time that you know works
negative control- should NOT show up on a gel; can be designed by not adding DNA or an enzyme to PCR and adding water instead
Understand how agarose gel electrophorese is used to analyze PCR results
used to separate DNA by size
-smaller migrate faster (closer to bottom)
-larger closer to top (closer to top)
if nothing shows up on gel then something went wrong in the experiment but if a positive and negative shows up then you missed something
Understand what the purpose of RT-PCR is and how you set up an appropriate RT-PCR reaction
goal is to look at changes at the RNA levels and is a way to quantitate if you have different amounts; isolating RNA from the cells then adding primer and enzyme to convert to DNA then PCR
RT
reserve transcriptase
Explain why RT-PCR is semi-quantitative and how agarose gel electrophorese is used to analyze the results
semi-quantitative because it is relative measures of RNA levels rather than definitive quantities and compares the expression levels of a gene to a loading control; agarose gel is used to analyze the results through separation of the DNA fragments, visualization, band intensity, and size confirmation
Explain how you set up a negative and positive control in a RT-PCR
Negative control - Do not add any RT enzyme and if there is a product that shows up, there is DNA contamination and you cannot trust the results. If nothing shows up, it is pure
Positive control - a sample that has worked multiple times. If it does not work there is something wrong with the reaction. The positive control ensures that the reaction conditions are suitable for amplification and that the target gene is indeed expressed in your sample.
Explain why you need to set up a RT-PCR with a primer for a housekeeping gene as a loading control
primer is used to translate RNA into DNA so that it can undergo PCR; housekeeping gene as a loading control is used as a comparison to the target gene so that the data can be normalized, it helps account for variations in RNA quantity and quality between samples to ensure that the changes are due to biological changes and not technical error
Explain how using the Ct value you can quantitate differences in RNA levels between two samples using qRT-PCR
Using the Ct value can help you to quantitate differences in RNA levels between two samples using qRT-PCR since a lower Ct value indicates a higher initial quantity of RNA.
Pros of RT-PCR
Cheaper
Easier to perform
Can confirm the presence or absence of a specific RNA transcript
Results can be visualized through gel electrophoresis
Cons of RT-PCR
Primarily qualitative and may not accurately reflect the relative expression levels of genes
Not monitored in real time
Pros of qRT-PCR
Provides precise quantitative measurements of gene expression levels, (more accurate)
Monitored in real time
Can accommodate multiple samples and targets simultaneously, making it suitable for large-scale experiments
Cons of qRT-PCR
Expensive
More complex setup
Understand how a microarray is set up
Cell types → culture → RNA isolation → Reverse transcription and fluorescent tagging → Hybridization onto microarray
each circle represents a different cell, so each spot is to be examined one at a time, sample A is labeled one color and sample B is labeled another color, if no color at all than none is in the cell, if both colors show (as a mix) then both are present in the cell
the purpose of a microarray
be able to look at multiple things at a time; able to identify how genes are expressed in various conditions; essentially the same as real-time PCR
pros and cons of a microarray compared to qRT-PCR
microarray is able to compare multiple samples at once but is more expensive
Understand the purpose and steps of a western blot
purpose: look at changes in one specific protein
steps: total proteins extraction (SDS detergent added to make all the proteins negatively charged), gel electrophoresis, separated proteins, blotting, protein detection (through antibodies), visualization
what do antibodies bind to
one specific protein
Understand the role of the SDS and the β-Mercaptoethanol in the loading dye.
SDS - coats proteins to give them a negative charge, and denature proteins
β-Mercaptoethanol - breaks disulfide bonds that hold protein together, therefore denaturing the proteins
Understand how a SDS-PAGE separates proteins
separates proteins by size based on their molecular weight by using SDS to denature proteins and impart a uniform negative charge. When an electric field is applied, proteins migrate through the polyacrylamide gel, with smaller proteins traveling faster and larger proteins moving more slowly. The result is a size-based separation of proteins, allowing for their analysis or further processing
Understand the role of the primary and secondary antibodies in a western blot
Primary Antibody - only recognizes one specific protein and binds to it
Secondary Antibody - contains a fluorescent tag that will bind to the primary antibody
Understand that western blot is a semi-quantitative method to detect changes in protein levels
Protein levels are normalized to a housekeeping gene for reference
Explain why you need to set up a western blot with an antibody for a housekeeping protein as a loading control
Including an antibody for a housekeeping protein in a Western blot allows you to normalize the expression of your target protein, ensures equal sample loading, checks for uniform protein transfer, and improves reproducibility. It serves as an essential quality control to verify that any observed differences in protein expression are biologically meaningful and not due to experimental inconsistencies.
Understand how an ELISA is set up to quantitate the level of a protein
very similar to western blot but more quantitative, it looks for a specific protein using an antibody in a dish (the more it binds to the antibody, the more protein you have, and the more fluorescent signal you will get)
Compare the benefits and drawbacks of an ELISA compared to a western blot
Benefits: ELISA is quantitative while western blot is semi-quantitative
Drawbacks: ELISA requires more optimization and is more expensive
Understand how a co-IP experiment is set up to determine if two different proteins interact
you can detect interaction between different proteins through antibody binding
Understand the major limitation of a co-IP
major limitation is that it is indirect (so you know they are part of a complex but do not know if it is direct or indirectly associated) and A co-IP might not be able to capture short lasting protein interactions
Understand how FRET is used to study protein interactions
use of two proteins, add a fluorescent tag to both of them, if they react with one another there will be fluorescents (stronger the interaction the more fluorescents there will be) but no fluorescents means no interaction
What are the benefits and drawbacks of FRET
Benefit- shows interaction between two proteins, quantitative
Drawback- expensive, at times hard to design the right probes so may get some drawbacks
Describe how co-Immunofluorescence is used to study protein interactions
No interaction = no fluorescence
What are the benefits and drawbacks of using co-immunofluorescence to study protein interactions?
Benefit - in real time and more physiological
Drawback: expensive and you get background fluorescence
Understand how siRNA;s work to cause knockdown of gene expression
siRNA binds to the coding region of the RNA, which leads to degradation
Understand siRNA;s target the coding region of a target mRNA
siRNA binds to one target
How are siRNA’s designed to target?
siRNA's are designed to target a specific mRNA, but can have off-target effects
Understand different assays that can be performed to determine efficiency of siRNA knockdown
Each assay provides different insights into siRNA efficiency, and combining assays (like qPCR for mRNA and Western blotting for protein) gives a more comprehensive view of knockdown effectiveness.
Understand how miRNA are produced
they are self-produced
Understand regions of target mRNA, miRNA bind to
miRNA can bind to multiple RNAs
Understand what determines how a miRNA inhibits translation of it's target mRNA or if it leads to the degradation of its target mRNA
Perfect complementarity: degradation (seen by red and blue lining up perfectly; no RNA no protein)
Partial complementarity: inhibits translation (spaces or bumps in the red and blue)
What are different assays that can be used to determine knockdown efficiency of a miRNA
The assays include qPCR, western blotting, and microarray analysis
Be able to compare siRNA and miRNA
siRNA - artificially made, binds perfectly to one target/RNA, if it binds perfectly degradation occurs
miRNA - made in the nucleus, binds to multiple targets; if it binds perfectly degradation occurs
Understand how the guide RNA and the Cas9 enzyme in CRISPER work together to cut out a specific gene
guide RNA recognized the gene and cas9 enzyme cuts it out
What does the addition of a donor DNA to the guide RNA and Cas9 do?
you can replace a defective gene with a correct copy of a gene
What happens when CRISPER knockouts the expression of a gene compared to siRNA?
CRISPER knockout causes a permanent knockout by editing the gene; genomic level (DNA); irreversible
siRNA causes temporary knockout of a gene by degrading the mRNA, transcriptional level (RNA); can be reversed if siRNA is removed
Understand the assays used to determine if CRISPER knockout of a particular gene is successful
The assays include RT-PCR and western blotting
Understand how a CRISPER experiment can have off - target effects
it can cut out the wrong gene which is known as an off-topic effect
Understand the ethical implications of CRISPER
genetic enhancements and cosmetics rather therapeutic reasons