sequencing

Question 1

Sanger sequencing

Answer 1

• uses dNTPs (DNA synthesis monomers) and ddNTPs (terminating DNA strand synthesis)
• use one ddNTP at a time and add the other 3 dNTPS so only one base is stopped
• repeat for the other 3
• line each gel up and read across the bands to create the COMPLIMENTARY sequence
• for ease of labelling dye each ddNTP a diff fluorescence (better visualisation than p32)

Question 2

Next generation sequencing

Answer 2

• ultra sound breaks up fragments - each one stuck to a different surface (eg bead) and amplified on the surface by PCR
• add a base at a time like before with a coloured terminator and record which one –> colour change electrically recorded
• able to sequence millions of fragments per run
  a quicker more cost effective method, more precise

Question 3

nano pore sequencing

Answer 3

• helix unwound by enzyme and strand passed through a nanopore which is just the right size for the strand
  each base is different size so blocks the pore differently - the change in current from that is what’s measured
• determines the base sequence on that strand

Question 4

MPS (massively parallel sequencing)

Answer 4

uses tech to manage many sequences at once
has high sensitivity and generates accurate profiles

Question 5

SNP sequencing

Answer 5

quality and quantity of DNA determines type used
1. SNP micro array - 200ng, not good w/ degraded
2. Targeted kit: kintellegence - 50pg - 1ng
3. Whole genome sequencing - 50pg, good for degraded