Genetic Mutations and Next Generation Sequencing

lecture 4 pt 1 + 2

Variants

• a change in the organism w/ respect to the human genome as a reference

difference between variants and mutations

• a mutation does not take into account the reference genome, a variation does not necessarily yield a phenotypical change

SNPs

Single nucleotide polymorphisms (" SNPs ", pronounced "SNIPS") are DNA sequence variations that occur when a single nucleotide differs from the reference DNA sequence.

structural variants

large chromosomal chunks are substituted, deleted or re-arranged in some way

microarrays

• A microarray is a silicon chip with wells containing single-stranded *oligonucleotide probes**.

• It relies on DNA’s complementary base pairing, where fluorescently labelled cDNA from normal and tumour samples is flushed over the chip and binds to matching probes.

• The wells appear to change colour depending on how the labelled cDNA binds, allowing differences in gene expression or DNA sequences between normal and tumour samples to be detected.

sanger sequencing

The double-stranded DNA is first denatured by heat to produce single-stranded templates.

- A short primer binds to the template, and DNA polymerase begins synthesising a complementary strand.

- The reaction mixture contains normal nucleotides (dNTPs) and a small amount of dideoxynucleotides (ddNTPs), which lack the 3′ hydroxyl group needed for chain elongation.

- When a ddNTP is incorporated, it terminates DNA synthesis, producing fragments of varying lengths that each end at a specific base.

- Each ddNTP is fluorescently labelled with a unique colour for A, T, G, or C.

- The fragments are separated by capillary electrophoresis according to length, and a laser detector reads the fluorescent signals to determine the DNA sequence.

NGS key take aways

allows rapid sequencing of an entire genome or targeted regions by reconstructing DNA from millions of small fragments.

The main steps are:

1. Fragmentation of DNA (sonication) – The patient’s DNA is cut into small fragments to make it suitable for sequencing. Sonication, which uses high-frequency sound waves, is commonly used to shear DNA randomly into fragments of a desired size, producing manageable pieces for library preparation.

2. Library preparation & amplification – Adaptors (short known sequences) are attached to the fragments. Each fragment is then amplified, usually via PCR, to generate sufficient copies for detection.

3. Sequencing by synthesis – The sequencer reads DNA one base at a time for each fragment. Each base (A, T, G, C) is tagged with a distinct fluorescent or chemical label, allowing the machine to detect which base is incorporated at each step.

4. Signal detection & sequence reconstruction – The sequencer records the fluorescent signals (colours) for each incorporation. By combining the reads from millions of fragments, a consensus sequence is reconstructed for the entire sample.

5. Variant analysis – The patient’s reconstructed DNA sequence is compared to a reference genome to identify variants, such as SNPs, insertions, deletions, or structural changes, which may be relevant for disease or research.