DNA Sequencing Flashcards
List two DNA sequencing technologies that fall into each of the following generations:
(a) First generation
(b) Second generation
(c) Third generation
(a) First generation:
✔ Sanger sequencing
✔ Pyrosequencing
(b) Second generation:
✔ Illumina sequencing
✔ Ion Torrent sequencing
(c) Third generation:
✔ Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing
✔ Oxford Nanopore Sequencing
Differentiate between pyrosequencing and Sanger sequencing.
Pyrosequencing:
✔ Detects nucleotide incorporation by measuring the release of pyrophosphate.
✔ Provides faster results and is suitable for short-read applications.
✔ Uses luciferase to generate light signals for nucleotide detection.
Sanger sequencing:
✔ Uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis and requires gel or capillary electrophoresis to read the sequence.
✔ Produces longer reads but is slower compared to second-generation methods.
✔ Requires a separate sequencing reaction for each read.
Differentiate between Illumina sequencing and Oxford Nanopore Sequencing.
Illumina sequencing
✔ Uses reversible dye terminators for nucleotide identification.
✔ Provides high accuracy and short reads (up to 300 bp).
✔ Requires extensive library preparation and amplification.
Oxford Nanopore Sequencing
✔ Detects nucleotides as they pass through a nanopore, measuring changes in electric current.
✔ Can generate long reads (up to several kilobases) and real-time sequencing.
✔ Portable and can be used for field applications, but has a higher error rate compared to Illumina.
What reagents are required for Sanger sequencing?
(1) Template DNA: The DNA to be sequenced.
(2) Primers: Short sequences that anneal to the template DNA and initiate DNA synthesis.
(3) Deoxynucleotide triphosphates (dNTPs): The building blocks for DNA synthesis.
(4) Dideoxynucleotide triphosphates (ddNTPs): Chain-terminating nucleotides labeled with fluorescent dyes or radioactive markers.
(5) DNA Polymerase: An enzyme that synthesizes the new DNA strand.
(6) Buffer Solution: Maintains the optimal conditions for the DNA polymerase activity.
What is library preparation in the context of Illumina sequencing?
Library preparation involves converting a sample of DNA (or RNA) into a format that can be sequenced on an Illumina platform.
applications of DNA sequencing
✔ Identification of pathogens
✔ Identification of mutations/variants
✔ Genomic epidemiology [Tracking the spread and evolution of infectious diseases by analyzing genetic sequences from different populations or time points.]
✔ Forensic studies
✔ Paternity testing
✔ Pharmacogenomics [This field uses DNA sequencing to understand how genetic variations affect individual responses to medications, leading to personalized drug therapies.]
