sequencing Flashcards
1
Q
Sanger sequencing
A
- 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)
2
Q
Next generation sequencing
A
- 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
3
Q
nano pore sequencing
A
- 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
4
Q
MPS (massively parallel sequencing)
A
uses tech to manage many sequences at once
has high sensitivity and generates accurate profiles
5
Q
SNP sequencing
A
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