5 DNA sequencing

what is Sanger sequencing?

a method of determining the sequence of nucleotides in a section of DNA.

what did sanger introduce which allowed us to find the exact DNA composition of the sequence?

• dideoxynucleotide - ribonucleotide with removed Oxygen.

• it prevents polymerase from adding any more nucleotides to the DNA chain.

Explain how to isolate a DNA sequence of interest

• Primer binds to region of interest.

• Polymerase extends primer by adding complementary nucleotides.

• Reaction stopped with dideoxynucleotide, preventing further extension.

• Specific fluorescent dye used to identify terminated nucleotide.

• Extension products vary in length, terminated with dideoxynucleotides.

outline how the extension products are separated by capillary electropheresis.

• DNA fragments move based on molecular weight.

• Laser excites dye-labeled DNA fragments in capillary.

• Detected light emissions interpreted by software for base call.

• Pool of DNA fragments measured and separated to obtain sequence.

Briefly explain how illumina sequencing works.

it works by simultaneously identifying DNA bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain

Briefly explain how Ion Torrent sequencing works.

• It works by detecting the release of H⁺ ions as nucleotides are incorporated into a growing DNA strand.

• When a nucleotide is added, it releases a H⁺ ion, which is detected by a sensor.

• The sequence of the DNA fragment is determined based on the order in which these ions are released during the sequencing process.

what is dNTP?

• deoxynucleotide triphosphate

• there are 4 types: adenine (dATP), cytosine (dCTP), guanine (dGTP), and thymine (dTTP)

essentially, __ change is used to determine how many bases were added with each cycle.

pH change.

note: the red is the DNA sequence.

Briefly explain how nanopore sequencing works.

It involves passing DNA through a nanopore & measuring changes in electrical current as individual nucleotides move through the pore. Each nucleotide produces a unique signal, allowing for real-time sequencing of DNA molecules.

summarise the 3 NGS methods.

• Illumina (uses fluorescent nucleotides)

• Ion Torrent (measures pH change)

• Nanopore (uses changes in electrical current through an artificial pore)

what is the purpose of the human genome project

to provide tools to understand:

• genetic factors in disease

• new strategies for treatment & prevention

what is the purpose of the 100,000 genomes project

• to move away from ‘one size fits all’ approach & more towards personalised healthcare.

• provides widespread sample of population to understand where variability is within normal (healthy & disease states)

what are medical applications of NGS?

identification of genetic diseases

what are pharma/biotech industry applications of NGS?

• new prognostics (predict predisposition to certain diseases)

• new diagnostics (see if a patient has a condition or not)

• Discovery of new targets for drug design

• Design of better medicines of the future