W8L11 - DNA Sequencing

Question 1

The 5 Phases of Sequencing

Answer 1

1. Maxam-Gilbert chemical cleavage DNA sequencing
2. Sanger sequencing using ddNTPs to terminate the reaction
3. Pyrosequencing
4. Next generation sequencing
5. Next-Next generation sequencing

Question 2

Next Generation Sequencing

Answer 2

Increases capacity to whole genome level
Combines several techniques together:
- amplification
- sequencing
- automation
Require either clonal amplification or direct molecule sequencing
All use an enzyme polymerase or ligase
Detection of sequence using either fluorescence or chemiluminescence

Question 3

Next-Next Gen Sequencing

Answer 3

‘Solid state’ detects the DNA as it moves through a nanopore or biological nanopore
- emission of fluorescence
- conductance of charge
Require minimal chemistry, no enzymes or nucleotides, inexpensive sample preparation
Extremely long reads theoretically possible

Question 4

Ion Torrent Sequencing

Answer 4

Nucleotide incorporates into DNA and this releases a H+ ion
Once this happens, ion sensors in the special plate will detect the slight change of pH
The change in pH read by computer and a signal is prodcued
If nucleotide isn’t a match then no H+ ion released
If two bases are incorporated then two H+ ions are released

Question 5

Challenges for Clinal Applications

Answer 5

Methodological challenges
Moving from monogenic to polygenic diseases
Uncertainty of results
The predicted frequency of recessive mutations in the population
- most people will contain one or more serious or lethal autosomal recessive mutation in a heterozygous state
Ethics