aPCR and DNA sequencing

What does qPCR stand for?

Quantitative Polymerase Chain Reaction.

What is the main difference between traditional PCR and qPCR?

qPCR measures DNA amplification in real time by detecting fluorescent signals, while traditional PCR measures DNA only at the end of all cycles.

What type of instrument is used in qPCR?

A thermal cycler capable of detecting fluorescence from the PCR reaction.

What does fluorescence indicate in qPCR?

It reflects the amount of amplified DNA; as DNA concentration increases, the fluorescence signal increases.

What is the principle behind TaqMan probe technology?

It uses a fluorescently labeled probe that binds to a specific DNA sequence between the primers. During extension, the probe is degraded by Taq polymerase, releasing a fluorescent signal proportional to the amount of target DNA.

Why is TaqMan technology highly specific?

Because the probe must perfectly match the target sequence to hybridize and generate a signal.

What does Ct stand for?

Ct stands for “Cycle threshold,” the number of cycles required for the fluorescent signal to exceed the background level.

What does a lower Ct value mean?

It indicates a higher amount of starting template (more mRNA or cDNA).

What does a higher Ct value mean?

It indicates less starting material (less mRNA or cDNA).

How can qPCR data be displayed

• As heat maps to show patterns of gene expression.

• As bar graphs showing Ct or ΔCt values on the y-axis.

What do heat maps illustrate?

They visually represent relative gene expression levels across samples, with color changes indicating up- or down-regulation.

What is normalization?

The process of correcting technical variability to reflect true biological differences.

Why is normalization important in qPCR?

It ensures that differences in gene expression reflect biological changes, not experimental inconsistencies.

What are common normalization strategies?

• To RNA concentration

• To tissue mass or cell number

• To reference (housekeeping) genes

• Using biological and technical replicates

• Applying statistical analysis to the data

What is a reference gene?

A gene with stable expression used as a baseline to normalize qPCR data.

What is a housekeeping gene?

A gene necessary for basic cell functions, often used as a reference gene.

What is the difference between a housekeeping gene and a reference gene?

All housekeeping genes can be reference genes, but not all reference genes must be housekeeping genes. The key concept is reference gene stability—a reference gene must be consistently expressed under experimental conditions.

What are the two main types of DNA sequencing methods?

• Sanger Sequencing – the original chain-termination method.

• Next-Generation Sequencing (NGS) – high-throughput, massively parallel sequencing technologies

Who developed the Sanger sequencing method?

Frederick Sanger.

What is the principle behind Sanger sequencing?

It uses chain-terminating dideoxynucleotides (ddNTPs) to stop DNA synthesis at specific bases, allowing sequence determination by fragment size analysis.

What scale is Sanger sequencing best suited for?

Small-scale projects such as sequencing individual genes or short DNA fragments.

What is Next-Generation Sequencing (NGS)?

Advanced sequencing technologies that allow simultaneous sequencing of millions of DNA fragments.

What is one common NGS platform?

Illumina sequencing.

What is an advantage of NGS over Sanger sequencing?

NGS is much faster, cheaper per base, and capable of sequencing entire genomes

What is a disadvantage of NGS?

It requires complex data analysis and produces shorter read lengths compared to Sanger sequencing.