Enda's real Question pool

Section A will be short answer style questions that examine recall & understanding. In this section there will be 15 questions worth 3 marks each (45 Marks total).

Describe what a microarray is and name two types of biological samples that can be analysed using this technology.

A microarray is a solid surface with thousands of probes that measure gene expression or genomic variation. You analyse mRNA from tissues or cells. You also analyse genomic DNA from blood or tissue biopsies.

Explain the purpose of oligonucleotide probes on a microarray.

They bind to specific sequences in the sample during hybridisation. This allows the system to detect which genes or SNPs match the probe sequence.

What is the function of Cy3 and Cy5 fluorophores in gene expression microarrays?

They label cDNA from two samples. Cy3 emits green light. Cy5 emits red light. The scanner measures their signal to compare expression between conditions.

Define “hybridisation” in the context of microarrays.

It is the binding of labelled sample DNA or cDNA to complementary probes on the chip through base pairing.

During scanning, a spot on a gene-expression microarray appears yellow. What does this indicate?

Both fluorophores show similar intensity at that probe. The gene shows similar expression in both samples.

What processing steps occur after scanning to convert the microarray image into usable data?

You subtract background noise. You quantify spot intensity. You normalise the signals. You convert the values into expression ratios.

What is a SNP and why does SNP analysis require knowledge of upstream and downstream genomic sequences?

A SNP is a single base difference at one position in the genome. You need flanking sequence to design probes or primers that bind the correct region.

State the role of whole genome amplification in SNP microarray analysis.

It increases the amount of DNA so the hybridisation step has enough material.

In SNP genotyping, what is the purpose of incorporating ddNTPs during PCR?

ddNTPs stop DNA synthesis at the SNP position. This allows detection of the base present at that site.

List two advantages of microarray technology that make it useful in precision medicine.

It analyses thousands of markers in one run. It shows expression or genotype patterns linked to disease risk or drug response.

Describe the general structure of a microarray chip and explain what is meant by a “cluster” or “bead.”

The chip has a glass or polymer slide with ordered spots that contain probes. A cluster or bead is a location with multiple copies of one probe, which improves signal strength.

What is the purpose of converting mRNA to cDNA during gene expression analysis?

cDNA is more stable. It allows fluorescent labelling and hybridises to DNA probes.

State what information can be obtained from a microarray scan showing intense green signals at certain spots.

Expression is lower in the test sample and higher in the control sample for those genes.

Explain why control samples (e.g., healthy tissue) are required in microarray experiments.

They offer a baseline. They show whether expression changes link to treatment or condition.

Define “background intensity” and explain why it must be subtracted during image processing.

It is signal from non-specific fluorescence on the chip. Removing it stops false increases in expression.

What is whole genome fragmentation, and why is it important in SNP or CNV microarray workflows?

You break DNA into smaller pieces. This allows equal hybridisation and avoids bias from high molecular weight DNA.

What key characteristic of SNPs makes them suitable for microarray-based genotyping?

They involve a single base difference that you detect with sequence-specific probes.

Why are unique dyes integrated with ddNTPs in SNP microarray PCR reactions?

The dye shows which base terminated the strand. This identifies the SNP genotype.

What is the purpose of using bioinformatics before designing primers for qRT-PCR?

You identify exon boundaries. You avoid secondary structure. You confirm unique binding sites.

Name two factors that must be considered when designing primers.

Melting temperature. GC content.

Define a housekeeping gene and give one example.

A housekeeping gene shows stable expression across conditions. GAPDH is an example.

Why do we measure mRNA levels when studying a disease model?.

You see how treatment or disease alters gene expression.

List a source of variation that housekeeping genes help to correct for.

Differences in RNA input between samples.

In your own words, explain what a Ct value is.

It is the cycle number where fluorescence rises above background during qPCR.

Describe the difference between the control group and treatment group in a qRT-PCR experiment.

The control group receives no treatment. The treatment group receives the drug or condition being tested.

Define a mutation and explain what makes it “permanent.”

A mutation is a change in DNA sequence. It becomes permanent when the cell replicates the altered sequence.

Distinguish between somatic and germline mutations.

Somatic mutations occur in body cells and do not pass to offspring. Germline mutations occur in sperm or egg cells and pass to children.

What is the difference between a point mutation and a chromosomal mutation?

A point mutation alters one base. A chromosomal mutation alters large sections or whole chromosomes.

Describe a silent mutation and explain why it does not alter protein function.

A silent mutation changes a base but keeps the same amino acid due to codon redundancy.

Compare conserved vs non-conserved missense mutations. Provide an example of the impact each may have.

A conserved change swaps an amino acid for one with similar properties so structure stays stable. A non-conserved change replaces it with an unrelated amino acid and disrupts folding or activity.

Explain the mechanistic difference between a transition and a transversion mutation.

A transition swaps a purine for the other purine or a pyrimidine for the other pyrimidine. A transversion swaps purine for pyrimidine or the reverse.

Explain which is more likely to occur, a transition and a transversion mutation.

Transitions occur more often because base chemistry favours same-class substitutions.

Describe the effect of UV light on DNA and why it can disrupt replication.

UV causes thymine dimers. These block polymerase movement.

What is a nonsense mutation, and why does it often result in a non-functional protein?

A nonsense mutation introduces a stop codon. Translation stops early and forms a short peptide.

Define an indel and explain the difference between a microindel and macroindel.

An indel is insertion or deletion. A microindel is a small change of a few bases. A macroindel removes or adds large regions.

What is a frameshift mutation, and how does it differ from an in-frame mutation?

A frameshift alters the reading frame and changes every codon downstream. An in-frame mutation removes or adds multiples of three, keeping the frame intact.

Describe the role of the mismatch repair (MMR) system.

It detects base-pair mismatches and fixes them after replication.

What type of nucleic acid does qRT-PCR measure?

It measures mRNA levels through cDNA.

Give one reason why RNA quality may vary between samples.

RNases degrade RNA during extraction.

What does a difference of 1 Ct cycle indicate in terms of gene expression?

A one cycle difference shows a twofold change in expression.

List the main steps in extracting mRNA from experimental samples.

Lyse cells. Bind RNA to a column or beads. Wash contaminants. Elute purified RNA.

Section B – Examines a student’s ability of application and analysis based off the content covered in the semester. In this section there will be 4 Questions presented and students will have to answer 3 which are worth 10 marks each (30 Marks total).

Compare gene-expression microarrays and SNP genotyping microarrays. Include:

• What each measures

• The sample preparation differences

• Colour interpretation differences

Gene-expression microarrays measure mRNA levels. SNP microarrays measure genotype at fixed positions. Expression uses mRNA converted to cDNA then labelled with dyes. SNP analysis uses genomic DNA that is fragmented and amplified. Expression arrays use red and green dye ratios to show up or down regulation. SNP arrays assign genotype based on dye signals linked to base identity.

Analyse the following scenario:

A microarray heat map shows consistent green signals for a set of 50 genes in a treated cell line compared to the control.

Explain:

(a) What this colour pattern suggests

(b) What experimental validation method should follow and why

a. This pattern shows reduced expression in treated cells.

b. You follow with qRT-PCR to confirm expression changes because it offers higher accuracy.

c. The treatment might suppress a pathway or block transcription factors for those genes.

You are comparing treated vs untreated cell lines using microarrays. The processed data show that signal intensity for a set of genes is consistently lower in treated samples.

Interpret this finding and describe two possible biological reasons for the observed under-expression.

This shows downregulation of these genes. Possible reasons include reduced transcription after drug exposure. Another reason is mRNA degradation due to stress.

Explain how primer self-annealing or hairpin formation can negatively impact a qRT-PCR experiment.

Primers bind to themselves instead of the target. This lowers amplification efficiency and gives inaccurate Ct values.

Why is it best practice to use multiple housekeeping genes when analysing gene expression?

One housekeeping gene might shift with treatment. Using several offers stable normalisation.

Explain why Ct values from different samples cannot be directly compared without normalisation.

.Each sample has different RNA input and efficiency. Normalisation removes these differences.

Explain how alkylating agents induce mutations and how these can lead to GC→AT transitions.

Alkylating agents add groups to guanine. The altered guanine mispairs with thymine during replication. This converts GC to AT in the next cycle.

A mutation results from improper repair of a double-strand break. Describe how NHEJ can create insertions, deletions, or translocations.

NHEJ creates insertions, deletions or translocations.

NHEJ joins broken ends with minimal checking of homology. Small base loss or addition occurs at the junction. Misjoining distant ends causes translocations.

Compare the DNA damage caused by ionising radiation to that caused by UV radiation.

Ionising radiation breaks DNA strands and forms reactive species. UV forms pyrimidine dimers that block polymerase

Explain slipped strand mispairing and describe how it leads to insertions or deletions in repeat-rich regions.

Repeat regions misalign during replication. The polymerase inserts extra repeats or skips repeats. This creates insertions or deletions.

Section C – Examines a student’s ability of knowledge synthesis and integration of knowledge based off the content covered in the semester. In this section there will be 2 Questions presented and students will have to answer 1 which are worth 25 marks (25 Marks total).

Construct a detailed experimental plan for identifying gene expression differences between two cancer subtypes using microarrays. Include:

• Sample preparation

• Labelling strategy

• Hybridisation and washing

• Scanning and signal extraction

• Statistical analysis

• Interpretation via heat maps

• Downstream validation steps

You begin with sample collection from both cancer subtypes. You extract high quality RNA with column or bead based kits. You assess integrity using electrophoresis or a bioanalyser. You convert equal RNA input to cDNA. You label each group with Cy3 or Cy5. You fragment and purify the labelled cDNA.

You place the labelled samples on the microarray slide. You allow hybridisation in a controlled chamber. You wash the slide to remove unbound fragments. You scan with a laser scanner. You collect red and green intensities for each probe.

You process the raw image by subtracting background. You quantify each spot. You normalise using methods such as global normalisation. You calculate fold changes and log ratios.

You create a heat map to view patterns across subtypes. You cluster genes that behave together. You identify expression signatures that separate the subtypes.

You validate key expression changes with qRT-PCR. You design primers for selected targets. You normalise to multiple housekeeping genes. You analyse fold change with the delta delta Ct method.

You interpret the combined results to define pathways that differ between cancer subtypes.

Describe how you would combine SNP microarrays to develop a complete diagnostic profile of disease-causing SNPs. Your answer should address:

• What each technology contributes

• How samples should be processed

• How conflicting or ambiguous results would be resolved

• Key limitations for clinical decision-making

Propose a full experimental plan using qRT-PCR to test whether a drug of interest alters expression of two target genes. Include controls, primer selection, housekeeping strategy, and data analysis.

Proposed experimental plan

1. Experimental groups and overall design

Use a controlled cell culture or tissue model.

Include at least three biological replicates per group.

Groups:

• Untreated control

• Vehicle control (same solvent as drug)

• Drug treated at one or more concentrations

Expose cells for a defined period that matches expected transcriptional response, for example 6 to 24 hours, based on prior data or pilot experiments.

Primary readout: expression of the two target genes compared with controls.

2. Sample collection and RNA extraction

After treatment, rapidly place culture on ice.

Wash cells with cold PBS to remove medium.

Lyse cells in RNA lysis buffer containing strong chaotropic salts and RNase inhibitors.

Extract total RNA using a column based or bead based kit following a standard protocol.

Include an on column DNase digestion step to remove residual genomic DNA.

Measure RNA concentration and purity by spectrophotometry.

Assess RNA integrity by gel electrophoresis or a microfluidic system.

3. cDNA synthesis

Use equal amounts of RNA from each sample, for example 500 ng to 1 µg.

Prepare a reverse transcription mix with random hexamers or oligo dT primers, dNTPs, reverse transcriptase, and buffer.

Include a no reverse transcriptase control for at least one sample to monitor genomic DNA contamination.

Run the reverse transcription reaction under optimal conditions for the chosen enzyme.

Dilute cDNA to a standard working concentration for qPCR.

4. Primer selection for target genes

Select gene specific primers for each target gene.

Use bioinformatics tools to choose primer pairs that:

• Span exon exon junctions where possible, to reduce genomic DNA amplification

• Have melting temperatures within a narrow range, for example 58 to 62 °C

• Show GC content within a moderate range, for example 40 to 60 percent

• Avoid strong secondary structure, primer dimers, and hairpins

Check in silico that each primer pair is unique in the genome.

Design amplicon length between 70 and 200 base pairs for efficient qPCR.

Order primers and validate efficiency on a standard dilution series of cDNA.

Accept primer sets with amplification efficiency between about 90 and 110 percent and with a single peak in melt curve analysis.

5. Housekeeping gene strategy

Select at least two candidate housekeeping genes that show stable expression in the cell type and under the treatment conditions, for example GAPDH, ACTB, HPRT1, or RPLP0.

Do not assume stability. Test housekeeping candidates across untreated, vehicle, and drug treated samples in a pilot qPCR run.

Evaluate Ct variation and amplification efficiency for each candidate.

Discard housekeeping genes with large Ct shifts across conditions or poor efficiency.

Use the most stable one or two housekeeping genes for normalisation.

If two or more housekeeping genes pass stability testing, use the geometric mean of their expression for normalisation.

6. qRT PCR reaction setup

Prepare reactions in a 96 well or 384 well plate, using a SYBR Green or probe based qPCR master mix.

For each sample and each gene (two targets plus housekeeping genes), set up technical triplicates to reduce pipetting variance.

Typical reaction mix per well:

• qPCR master mix

• Forward and reverse primers at optimal concentration, for example 200 to 500 nM

• cDNA template at constant amount across wells

• Nuclease free water to volume

Include no template controls for each primer pair to detect contamination or primer dimers.

Seal plates and briefly centrifuge to remove bubbles.

7. qRT PCR run and quality checks

Run qPCR on a real time thermocycler with an initial activation step, then 35 to 40 cycles of denaturation and annealing/extension at the chosen temperatures.

For SYBR Green assays, perform a melt curve at the end to confirm a single specific product.

Check amplification plots for smooth, sigmoidal curves and similar baseline across wells.

Exclude wells with irregular amplification, outlier Ct values among triplicates, or evidence of primer dimers in no template controls.

8. Data processing and Ct analysis

For each gene and each sample, average the Ct of technical triplicates.

Confirm that housekeeping gene Ct values show minimal variation between groups.

For each sample, calculate ΔCt for each target gene:

ΔCt = Ct(target gene) − Ct(reference housekeeping gene or geometric mean of housekeeping genes)

Select a reference group, usually untreated control.

For each sample, calculate ΔΔCt:

ΔΔCt = ΔCt(sample) − mean ΔCt(reference group)

Convert to fold change using the 2^−ΔΔCt method.

Values above 1 indicate relative upregulation, values below 1 indicate relative downregulation.

9. Statistical analysis

For each gene, work with ΔCt values for statistical testing, because these values follow a more normal distribution than fold change.

Use appropriate tests, for example a t test for two groups or one way ANOVA for multiple conditions, followed by post hoc tests if needed.

Assess significance level, for example p less than 0.05.

Report mean fold change plus standard deviation or standard error for each group.

10. Controls and interpretation

Use untreated control as baseline expression.

Use vehicle control to separate effects of the solvent from effects of the drug.

Confirm absence of amplification in no template controls.

Confirm high Ct values or no amplification in no reverse transcriptase controls, which indicates minimal genomic DNA contamination.

Confirm single peaks in melt curves for SYBR Green assays, which supports specificity.

If the drug produces significant changes in expression for both target genes relative to controls, and housekeeping genes remain stable, conclude that the drug alters transcription of the target genes under the tested conditions.

Describe a major mechanism by which duplications arise. Evaluate how each mechanism can contribute to genetic disorders.

Evaluate how regulatory element mutations (promoter, splice-site, poly-A tail) can impact gene expression and protein function.

Regulatory element mutations alter when, where, and how much RNA and protein the cell produces.

Promoters, splice sites, and poly A signals each control a different step.

Changes in these regions often lead to disease through loss or misregulation of protein function.

1. Promoter mutations

Promoters control transcription initiation by RNA polymerase and binding of transcription factors.

a) Reduced promoter activity

A mutation in a core promoter motif, for example TATA box or initiator element, lowers affinity for transcription machinery.

Transcription starts less frequently.

mRNA levels fall.

Protein levels fall.

For gene