DNA sequencing

Question 1

The most commonly used foundational method of DNA sequencing is known as

Answer 1

dideoxy chain-termination sequencing or Sanger sequencing.

Question 2

DNA sequencing

Answer 2

Ultimate way of characterising DNA – at single nucleotide resolution!

Question 3

Maxam-Gilbert

Answer 3

– chemical degradation (redundant)

Question 4

Sanger dideoxy chain termination

Answer 4

Enzymatic synthesis
Automated Sanger sequencing (capillary)

Question 5

How is DNA sequenced by the Sanger method (Chain-termination)?

Answer 5

1. DNA is mixed with a primer complementary to the target DNA, along with DNA polymerase, and the four deoxyri-bonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP).
2. Key is the inclusion of a small amount of modified deoxyribonucleotides, called dideoxynucleotides (ddNTPs).
3. ddNTPs lack the 3′ oxygen required to form a phosphodiester bond with another nucleotide.
4. If DNA polymerase incorporates a ddNTP (c.f. dNTP) into a synthesis chain then, the synthesis will terminate at that base pair.
5. DNA polymerase will randomly incorporate ddNTPs as it elongates the template DNA. When this occurs, synthesis stops.
6. Eventually a ddNTP will be inserted at every location in the sequence of newly synthesized DNA molecules so that each strand synthesized differs in length by one nucleotide and is terminated by a ddNTP.
7. These fragments can then be separated to obtain the complete sequence of the DNA.
8. Original method used radioactive ddNTPs and fragments were separated on large gels

Question 6

Slide 17. Chapt 20 -Part 2 - 1:32 seconds

Question 7

Question 8

Modern Sanger sequencing is automated

Answer 8

1. Fragments are now separated in an ultrathin-diameter polyacrylamide tube gel called a capillary gel.
2. Fragments are scanned with a laser, stimulating fluorescent dyes on each DNA fragment (different wavelength for each ddNTP).
3. Light emission is captured by a detector and the computer assembles the entire DNA sequence into an output format called an electropherogram.
4. Such platforms were essential to the progress of the Human Genome Project

Question 9

Answer 9

An electropherogram

Question 10

Next Generation Sequencing Technologies – 454 sequencing

Answer 10

Demand for sequencers that are capable of generating millions of bases of DNA sequences in a relatively short time.
The first NGS instruments produced as much data as 50 capillary electrophoresis systems and were up to 200 times faster and cheaper than conventional Sanger approaches.

Question 11

Next Generation Sequencing (NGS)

Answer 11

Massive parallel sequencing
- Roche 454 FLX (pyrosequencing)
- ABI SOLiD 5500 (sequencing by ligation)
- Ilumina HiSeq (sequencing by synthesis)
Single molecule sequencing (Third-Generation Sequencing)
- PacBio SMRT
- Oxford Nanopore (MinION)

Question 12

Slide 20 - 22. Chapt.20 - Part 2

Question 13

Pyrosequencing - 454

Answer 13

First NGS technology to be commercialised (2005) by 454 Life Sciences
Beads are bound to fragmented genomic DNA
PCR amplified and added to wells (mixed with DNA Pol)

Question 14

Pyrosequencing:
Steps/process

Answer 14

A single, labelled nt (e.g. dATP) is flowed over each well
Each well has a bead with primers attached to the DNA
When a complementary nt passes template, adjacent to primer, it is added to the 3’ end of the primer by DNA Pol
Releases pyrophosphate – chemiluminescent reaction (light produced by firefly enzyme, luciferase)
Emitted light is recorded
Generate read lengths of 400bp, 400Mbp/10 hr run

Question 15

Pyrosequencing:
Steps/process

Question 16

Sequencing by synthesis - Illumina

Answer 16

Library construction
> Fragment, attach adapter DNA

Cluster generation
> Add to flow cell
> Hybridisation to anchored oligos
> Bridge amplification

Sequencing
> Single fluorescently labelled base incorporated at a time
> Imaging of one of four fluorophores
> Unbound fluorophores are washed away and another dNTP is washed over
the flow cell

Data analysis
> Sequencing occurs for millions of clusters at once
> Images transformed into basecalls and ‘reads’

Question 17

https://youtu.be/fCd6B5HRaZ8

Question 18

Single molecule real-time sequencing - PacBio

Answer 18

A single molecule of DNA Pol is anchored in a nanowell (10nm)
ssDNA template is flooded and a single molecule is bound by the DNA Pol
Four nucleotides are labelled fluorescently and are incorporated in the growing strand by DNA Pol
Individual nucleotides generate a pulse of colour when the fluorescent dye is clipped off during incorporation
Read-lengths of >10 kb

Question 19

Nanopore sequencing – Oxford Nanopore

Answer 19

The four nucleotides produce different ionic currents when a DNA molecule moves through a nanopore – can be detected
Allows sequencing of a single molecule of DNA or RNA without the need of PCR or dNTPs or any sort of chemical labelling.
Membrane with a single nanometer-sized protein pore (nanopore) separates two wells containing an electrolyte.
The enzyme present in nanopore unwinds the DNA strands (just like helicase) and provides a passage for the ssDNA to enter the nanopore channel.
When voltage is applied, the ions of the solution passes through the nanopore along with the DNA strand.

Question 20

Advantages of Nanopore method:

Answer 20

Long contigs - reads from 50 to 100 kb can be sequenced
It is cheaper, costing $750 per sequencing
Does not require any amplification (PCR), no dNTPs, or chemical labels

Question 21

Disadvantages:

Answer 21

High error rate