What does the central dogma of molecular biology state?
Genetic information flows from DNA → RNA → protein, and DNA can accumulate mutations.
What is meant by multi-omics?
Integration of genome, epigenome, transcriptome and other data layers to understand biology and disease.
Why has DNA sequencing become central to modern medicine?
It enables detection of disease-causing variants, cancer mutations, and population-scale genetic patterns.
What major technological shift occurred around 2007?
The introduction of next-generation sequencing (NGS).
Why is reducing sequencing cost important?
It allows large-scale clinical and population genomics projects.
What is the purpose of PCR?
To amplify a specific DNA region.
Which components are required for PCR?
Template DNA, forward and reverse primers, dNTPs, DNA polymerase, MgCl₂ buffer.
What is the principle of Sanger sequencing?
Chain termination using dideoxynucleotides (ddNTPs).
Why do ddNTPs terminate DNA synthesis?
They lack a 3′ OH group required for chain extension.
Why is Sanger sequencing considered the gold standard?
It has very high accuracy and a very low error rate.
What is a limitation of Sanger sequencing in cancer genomics?
It cannot reliably detect low-frequency somatic mutations.
What defines next-generation sequencing?
Massively parallel sequencing of millions of DNA fragments.
What type of reads does Illumina sequencing generate?
Short reads (50–300 bp).
What does sequencing by synthesis mean?
Bases are identified as they are incorporated during DNA synthesis.
What is a reversible terminator nucleotide?
A fluorescently labelled nucleotide that temporarily blocks further extension.
Why are fluorescent dyes cleaved after each cycle?
To allow incorporation of the next nucleotide.
What is a flow cell in Illumina sequencing?
A glass slide coated with oligonucleotides that capture DNA fragments.
What is bridge amplification?
PCR amplification of fragments directly on the flow cell to form clusters.
What is multiplexing in NGS?
Sequencing multiple samples together using index sequences.
What is depth of coverage?
The number of reads covering a genomic position.
Why is high coverage important?
It improves accuracy and variant detection sensitivity.
What limits short-read sequencing?
Difficulty detecting large structural variants and repetitive regions.
What are paired-end reads?
Reads obtained from both ends of a DNA fragment.
Why are paired-end reads useful?
They improve mapping accuracy and help detect structural variants.
