Seminar Quiz 10/29/24

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?

1. DNA fingerprinting

2. Classification of organisms

3. Genotyping

4. Prenatal diagnosis

5. Mutation screening

6. Drug discovery

7. Bioinformatics

8. Genomic cloning

9. Human Genome Project

10. Site-directed mutagenesis

11. 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

1. Cheaper

2. Easier to perform

3. Can confirm the presence or absence of a specific RNA transcript

4. Results can be visualized through gel electrophoresis

Cons of RT-PCR

1. Primarily qualitative and may not accurately reflect the relative expression levels of genes

2. Not monitored in real time

Pros of qRT-PCR

1. Provides precise quantitative measurements of gene expression levels, (more accurate)

2. Monitored in real time

3. Can accommodate multiple samples and targets simultaneously, making it suitable for large-scale experiments

Cons of qRT-PCR

1. Expensive

2. 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.

1. SDS - coats proteins to give them a negative charge, and denature proteins

2. 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

1. Primary Antibody - only recognizes one specific protein and binds to it

2. 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

1. Benefits: ELISA is quantitative while western blot is semi-quantitative

2. 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

1. Benefit- shows interaction between two proteins, quantitative

2. 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?

1. Benefit - in real time and more physiological

2. 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