Expression Level Normalization Methods in Cell Biology Experiments Quiz

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Questions and Answers

Which scientist coined the term 'plasmid'?

James Watson

Francis Crick

Gregor Mendel

Joshua Lederberg (correct)

What is the function of plasmids in bacteria?

To provide antibiotic resistance

To carry genes that benefit the survival of the organism (correct)

To replicate independently of chromosomal DNA

To exchange genes through bacterial conjugation

What is the copy number of plasmids in a single cell?

Always one

Four

Varies from one to several hundreds (correct)

Depends on the size of the cell

What is the purpose of CsCl density gradient centrifugation in isolating plasmids?

To separate chromosomal DNA from plasmid DNA Signup and view all the answers

Which of the following is a hazardous and volatile chemical used in the PCR experiment?

Phenol Signup and view all the answers

What is the purpose of using a carrier like glycogen in the precipitation step of the PCR experiment?

To aid in the washing process Signup and view all the answers

Which temperature is typically used to denature DNA molecules in PCR?

95oC Signup and view all the answers

What is the purpose of site-directed mutagenesis in molecular biology?

To make intentional changes to a gene's DNA sequence Signup and view all the answers

Which technique is used to assess whether real-time PCR assays have produced single, specific products?

Melting curve analysis Signup and view all the answers

What is the temperature at which 50% of DNA is denatured known as?

Melting point Signup and view all the answers

What is the process of synthesizing single-stranded DNA using single-stranded RNA as a template called?

Reverse transcription Signup and view all the answers

What is the Ct value in real-time PCR?

The number of cycles it took to detect a real signal above background fluorescence Signup and view all the answers

Which of the following is a disadvantage of qPCR?

Expensive equipment Signup and view all the answers

What is the formula to calculate fold change in gene expression using qPCR?

Fold change = Cttarget - Cthousekeeping Signup and view all the answers

What is the purpose of adding a pH indicator in colorimetric LAMP?

To enhance visual detection of products Signup and view all the answers

What is the principle behind pyrosequencing?

Detection of pyrophosphate release Signup and view all the answers

Which of the following methods can be used to break apart cells and release their inner contents?

Treatment with an enzyme that has cell lytic properties Signup and view all the answers

What is the purpose of separating DNA by Cesium Chloride?

To separate DNA from RNA and protein based on their buoyant density Signup and view all the answers

What happens to RNA during the separation process using Cesium Chloride?

RNA sinks to the bottom of the tube Signup and view all the answers

What is the purpose of loading a sample mixed with Cesium Chloride into an ultracentrifuge?

To separate the different macromolecules in the sample Signup and view all the answers

Which of the following is a reason why continued thermal cycling does not lead to the production of significant amounts of product anymore?

Depletion of the reagents Signup and view all the answers

What is the purpose of using a fluorescent marker in real-time PCR?

To monitor the formation of products as the reaction proceeds Signup and view all the answers

Which detection method in real-time PCR relies on the intercalation of a dye with the DNA double helix?

Detection Method 1: Intercalating dye (e.g. SYBR green) Signup and view all the answers

How does the hydrolysis probe technique in real-time PCR generate fluorescence?

The probe is cleaved by the polymerase enzyme, separating the reporter and quencher molecules Signup and view all the answers

Which enzyme is responsible for degrading nucleotides in the reaction mixture?

Apyrase Signup and view all the answers

Which substrate is added to the reaction mixture along with Adenosine 5’ phosphosulfate (APS)?

Luciferin Signup and view all the answers

Which nucleotide is added to the reaction mixture in pyrosequencing?

dATP Signup and view all the answers

What is released when DNA polymerase catalyzes the incorporation of a complementary nucleotide?

Pyrophosphate (PPi) Signup and view all the answers

What is the limitation of pyrosequencing when it comes to detecting homopolymeric regions?

Homopolymeric regions longer than 10 bases cannot be resolved Signup and view all the answers

What triggers a series of enzymes in pyrosequencing?

The release of pyrophosphate (PPi) Signup and view all the answers

Study Notes

Plasmids and Their Functions

The term 'plasmid' was coined by American geneticist Joshua Lederberg.
Plasmids function primarily as vehicles for gene cloning in bacteria, allowing for the transfer of genetic material.
The copy number of plasmids in a single bacterial cell can range from a few to several hundred.

CsCl Density Gradient Centrifugation

CsCl density gradient centrifugation is used to isolate plasmids by creating a gradient where plasmids can be separated based on their buoyant density.
During this process, RNA tends to form a separate layer and is effectively separated from DNA.

Hazardous and volatile chemicals used in PCR include substances like dimethyl sulfoxide (DMSO).
Glycogen is used as a carrier in the precipitation step of PCR to increase the yield of DNA during extraction.
The denaturation temperature for DNA molecules in PCR is typically around 94-98°C.

Site-Directed Mutagenesis

Site-directed mutagenesis allows for the precise modification of specific DNA sequences to study gene function.

Real-Time PCR

Real-time PCR is assessed for producing single, specific products using melting curve analysis.
The temperature at which 50% of DNA is denatured is termed the melting temperature (Tm).
The Ct (threshold cycle) value indicates the cycle number at which fluorescence exceeds a threshold level, reflecting the quantity of target DNA.

qPCR Limitations

A common disadvantage of qPCR is its potential for non-specific amplification and inaccurate quantification.

Gene Expression Analysis

Fold change in gene expression using qPCR can be calculated using the formula: Fold Change = 2^(ΔCt).
A pH indicator is used in colorimetric LAMP to reveal the presence of target DNA through a color change.

Pyrosequencing

Pyrosequencing relies on the release of pyrophosphate during DNA synthesis to generate a light signal for nucleotide detection.
A limitation of pyrosequencing is its difficulty in accurately detecting homopolymeric regions due to signal saturation.

Enzyme Activity in Reactions

In the reaction mixture, nucleotides are degraded by enzymes such as exonucleases.
Adenosine 5’ phosphosulfate (APS) is typically used alongside the nucleotide substrate in processes like pyrosequencing.

Centrifugation and DNA Separation

Loading a sample mixed with CsCl into an ultracentrifuge allows for the separation of molecules based on density, resulting in purified plasmids or DNA.
Continued thermal cycling in PCR may yield minimal product due to depletion of reagents or accumulation of inhibitory byproducts.

Fluorescent Markers and Detection in PCR

Fluorescent markers in real-time PCR enable real-time monitoring of DNA amplification through emitted fluorescence during reactions.
The intercalation of a dye with DNA double helix is the principle of SYBR Green detection methods in real-time PCR.
The hydrolysis probe technique generates fluorescence as a result of the cleavage of a probe upon amplification of the target DNA.

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Description

This quiz tests your understanding of expression level normalization methods in cell biology experiments. Topics covered include Ct values, gene expression, normalization to GAPDH and ACTB, cancer cells, neuronal differentiation, and pluripotent cells.