sequencing Flashcards

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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)
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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
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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
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4
Q

MPS (massively parallel sequencing)

A

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

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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

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