Functional Genomics & Mutation Analysis Flashcards
What is a Luciferase Reporter Assay and how does it work?
A Luciferase Reporter Assay is used to measure promoter or enhancer activity by linking a regulatory DNA sequence to a luciferase gene and measuring light output.
✔ Steps:
1️⃣ Clone the promoter or enhancer of interest upstream of a luciferase reporter gene in a plasmid.
2️⃣ Transfect the plasmid into cells and incubate.
3️⃣ Lyse cells to extract luciferase protein.
-If the regulatory sequence is active, it drives luciferase expression.
4️⃣ Add a luciferin substrate → luciferase enzyme produces light, which is detected by a luminometer.
✔ Results Interpretation:
High luminescence = Strong promoter/enhancer activity.
Low luminescence = Weak or inactive regulatory sequence.
How can you use a Luciferase Reporter Assay to test the effect of a mutation in a promoter region?
Introduce a wild-type and mutant promoter into separate luciferase constructs.
Transfect both into cells and compare luminescence output.
If the mutation reduces luminescence, it suggests the mutation weakens promoter activity (e.g., loss of a transcription factor binding site).
What is an EMSA and what does it detect?
EMSA (Electrophoretic Mobility Shift Assay) is used to detect protein-DNA interactions.
1️⃣ Label the DNA probe
A short double-stranded DNA sequence (probe) is radioactively (32P) or fluorescently labeled so it can be detected.
This probe contains a specific sequence that a protein may bind to.
2️⃣ Incubate DNA with the protein of interest
Mix the labeled DNA probe with a purified protein or nuclear extract.
If the protein recognizes and binds to the DNA, a protein-DNA complex forms.
3️⃣ Run the mixture on a non-denaturing polyacrylamide gel
The gel is run under low salt conditions to maintain protein-DNA binding.
Free DNA moves faster, while protein-bound DNA moves slower (shifted band).
4️⃣ Visualize the results
Compare lanes with and without protein.
If a shift occurs (slower-moving band), it means the protein binds the DNA.
How would you determine if a mutation in a transcription factor binding site disrupts binding using EMSA?
Perform EMSA using wild-type vs. mutant DNA probes.
If the mutant probe fails to bind or shows a weaker shift, it means the mutation disrupts protein binding.
What is ChIP-Seq and what is it used for?
ChIP-Seq (Chromatin Immunoprecipitation Sequencing) identifies transcription factor binding sites and histone modifications across the genome.
✔ Steps:
1️⃣ Fix protein-DNA interactions
Treat live cells with formaldehyde to crosslink proteins to the DNA they are bound to.
This locks the transcription factors or histones in place.
2️⃣ Break the DNA into small pieces
Fragment the chromatin using sonication (sound waves) or enzymes to chop the DNA into short fragments (~200–500 bp).
3️⃣ Pull down protein-bound DNA using an antibody
Add an antibody that specifically recognizes your protein of interest (e.g., a transcription factor or a histone modification).
Use magnetic beads to isolate only the protein-DNA complexes.
4️⃣ Remove the proteins to extract the DNA
Heat the sample to reverse the crosslinks, separating the DNA from the proteins.
Purify the DNA to remove everything except the DNA fragments that were bound to the protein.
5️⃣ Sequence the DNA with Next-Generation Sequencing (NGS)
Perform high-throughput sequencing to determine the genomic locations of the protein binding sites.
✔ Results Interpretation:
Mapped sequences reveal where transcription factors or histone modifications occur in the genome.
Used for gene regulation studies, enhancer mapping, and epigenetics research.
How would you confirm if a transcription factor binds to a specific gene promoter using ChIP-Seq?
Perform ChIP using an antibody against the transcription factor.
Sequence and align the precipitated DNA to the genome.
If peaks appear at the gene promoter, it confirms binding in vivo.
What is qPCR and how does it measure gene expression?
qPCR (Real-Time PCR) quantifies gene expression levels by measuring fluorescent signals during DNA amplification.
✔ Steps:
1️⃣ Convert mRNA into cDNA using reverse transcription.
2️⃣ Amplify the target gene using specific primers.
3️⃣ Use fluorescent dyes (SYBR Green) or probes (TaqMan) to monitor real-time DNA amplification.
✔ Results Interpretation:
Ct value (Cycle threshold): The cycle at which fluorescence exceeds background levels.
Lower Ct value = Higher initial gene expression.
Higher Ct value = Lower gene expression.
How do you compare gene expression between two conditions using qPCR?
Use housekeeping genes (e.g., GAPDH, ACTB) for normalization.
Calculate fold-change using the ΔΔCt method.
Example: If a cancer gene shows a lower Ct value in tumor cells, it suggests overexpression in cancer.
What is a microarray and how does it work?
Microarrays measure gene expression levels or detect genetic variants (e.g., SNPs, copy number variations) using labeled DNA/RNA hybridization.
✔ Steps:
1️⃣ Isolate RNA and convert it into cDNA.
2️⃣ Label cDNA with fluorescent dyes.
3️⃣ Hybridize to a solid surface containing complementary DNA probes.
4️⃣ Measure fluorescent intensity to determine gene expression levels.
✔ Results Interpretation:
Red signal = Gene is upregulated.
Green signal = Gene is downregulated.
Yellow signal = No change in expression.
Minigene
Used to study RNA splicing and splice site mutations in a controlled system.
1️⃣ Clone gene region into a minigene vector (wild-type & mutant).
2️⃣ Transfect cells with the minigene constructs.
3️⃣ Extract RNA and perform RT-PCR to analyze transcript variants.
4️⃣ Compare wild-type vs. mutant splicing patterns (e.g., exon skipping, intron retention).
5️⃣ Interpret results to determine if the mutation affects splicing.
Application:
✔ Identifies splicing defects in disease-associated mutations.
✔ Confirms the functional impact of intronic and exonic variants.
