DNA Sequencing Flashcards
(24 cards)
What is DNA sequencing?
It is the process of determining the order of nucleotides (A, T, G, C) in a DNA molecule.
Why is DNA sequencing important in biology?
It helps in evolutionary studies, identifying gene mutations, diagnosing diseases, detecting pathogens, and confirming genetic experiments.
What are the four nucleotide bases in DNA?
Adenine (A), Thymine (T), Guanine (G), and Cytosine (C)
What are the main generations of DNA sequencing?
First Generation (Sanger), Second Generation (NGS), Third Generation (Long-read sequencing)
What is the principle behind Sanger sequencing?
Incorporation of chain-terminating ddNTPs during DNA replication, stopping extension at specific bases.
What are the steps of Sanger sequencing?
Denaturation & primer binding, extension with ddNTPs, fragment separation, visualization using fluorescence.
What is a key visual output of Sanger sequencing?
A chromatogram that shows fluorescence intensity for each base position.
Name one pro and one con of Sanger sequencing.
Pro: High accuracy; Con: Low throughput and expensive for large-scale sequencing.
What does NGS stand for and how is it different from Sanger sequencing?
Next-Generation Sequencing; it sequences millions to billions of short reads in parallel, unlike the single-fragment focus of Sanger.
What are the basic steps of NGS?
DNA extraction, library prep, bridge amplification, sequencing by synthesis, and data analysis.
What is bridge amplification in NGS?
It is the PCR-based step that amplifies DNA fragments into clusters on a flow cell.
What is a key limitation of NGS?
Short reads (50–500 bp), making it hard to assemble repetitive DNA regions.
What is a major advantage of NGS over Sanger?
High throughput and lower cost per base.
What is long-read sequencing?
Sequencing technology that reads long fragments (10–50 kb+) of DNA directly.
What are two long-read sequencing platforms?
PacBio (SMRT sequencing) and Oxford Nanopore Technologies (ONT)
How does PacBio SMRT sequencing work?
It uses circular DNA templates and records fluorescent signals in real time as bases are added.
How does Oxford Nanopore sequencing determine bases?
By detecting changes in ionic current as single-stranded DNA passes through a nanopore.
What are the main pros of long-read sequencing?
Very long reads, fast runs, easy assembly, epigenetic info retained, portable platforms.
What is a major con of long-read sequencing like ONT?
Higher error rate than NGS; needs higher coverage for accuracy.
What are common sources of error in DNA sequencing?
Poor DNA quality, degradation, contamination, and low DNA quantity.
When is Sanger sequencing most appropriate?
For small projects targeting 1–2 genes, when high accuracy is needed.
When is Illumina sequencing ideal?
For high-throughput needs and when DNA is fragmented or of low quality.
When should PacBio be used?
For generating high-quality reference genomes with accurate long reads.
When is Oxford Nanopore best used?
For portable, fast sequencing in the field and for population-scale projects.
