[W5] DNA Sequencing Flashcards
(30 cards)
What is DNA sequencing?
The process of determining the exact sequence of nucleotides (A, T, C, G) in a DNA molecule.
Which enzyme is central to sequencing-by-synthesis technologies?
DNA polymerase.
What are the two main types of DNA sequencing?
- Sanger (dideoxy) sequencing
- Next-generation sequencing (NGS)
What is required for Sanger sequencing?
- DNA template
- Primer
- DNA polymerase
- dNTPs (normal nucleotides)
- ddNTPs (chain-terminating nucleotides)
How do ddNTPs terminate DNA synthesis?
They lack a 3′-OH group, preventing further nucleotide addition.
What is the typical read length for Sanger sequencing?
Up to ~900 bp.
What are the strengths and weaknesses of Sanger sequencing?
- Strengths: High accuracy for long reads
- Weaknesses: Expensive and inefficient for whole genomes
What is the role of fluorescent tagging in automated Sanger sequencing?
Fluorescently labelled ddNTPs allow detection of terminated fragments during capillary electrophoresis.
How is NGS different from Sanger sequencing?
NGS allows massively parallel sequencing, is faster and cheaper, but produces shorter reads (50–500 bp).
What are the 3 general steps in NGS?
- Library preparation
- Template amplification (e.g. PCR)
- Sequencing
What is the purpose of adapter sequences in library prep?
Enable DNA to bind to sequencing chips and provide priming sites.
What is emulsion PCR (ePCR)?
PCR in tiny water-in-oil droplets; each drop contains a DNA molecule and functions as a microreactor for clonal amplification.
What is bridge PCR?
Amplification on a flow cell using oligos that form a ‘bridge’ between adaptor sequences and the surface, allowing repeated cycles of amplification.
How does Illumina sequencing work?
It uses DNA polymerase to incorporate reversibly fluorescent and terminator-modified nucleotides, which are imaged, then chemically reset for the next cycle.
What is a limitation of Illumina sequencing?
Increased error rate over time due to incomplete removal of fluorescent tags.
Name four types of NGS sequencing technologies.
- Pyrosequencing
- Sequencing by synthesis (e.g., Illumina)
- Sequencing by ligation
- Ion semiconductor sequencing
What is pyrosequencing?
Detects pyrophosphate release upon nucleotide incorporation, converting it to light via enzymatic reactions.
Why is sequence assembly needed in NGS?
NGS produces short reads that must be pieced together to reconstruct the full sequence.
What types of biological molecules can be analyzed using NGS?
- DNA
- RNA (transcriptomics)
- Proteins (indirectly via ribosome profiling)
Name five applications of DNA sequencing.
- Whole genome sequencing
- Targeted sequencing of genes/mutations
- Metagenomics
- Exome sequencing
- SNP identification
What is the exome?
All the exons in a genome — the coding regions (~1–2% of the genome).
What are the two steps in exome sequencing?
- Target enrichment
- Sequencing
Give an example of clinical use of whole-exome sequencing (WES).
WES corrected a misdiagnosis of Bartter syndrome by identifying a novel mutation in the SLC26A3 gene (actually congenital chloride-losing diarrhoea).
What are SNPs?
Single base changes in the genome; the most common type of genetic variation.
