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Flashcards in DNA sequencing future developments Deck (14)
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1
Q

5 phases of sequencing

A
  1. maxam-gilbert chemical cleavage DNA Seq: slow
  2. Sanger sequencing w/ ddNTPs = terminate rxn
  3. Pyrosequencing
  4. Next generation sequencing: multiple rxn per bead
  5. Next-next gen sequencing: detect DNA as move through nanopore
2
Q

disadvantage to pyrosequencing

A

rxn vol inc = efficiency dec

*from adding nucleotides for each rxn cycle

3
Q

what techniques are combined in Next gen sequencing

A
  • (cloning) Amplification (454 method)
  • (direct molecule) Sequencing (Helicos, pacific biosystem method)
  • Automation
4
Q

describe technique of next gen sequencing

A
  • all use polymerase or ligase

- detect sequence using fluorescence or chemiluminescence

5
Q

Describe the general amplification technique in next gen

A
  • water in oil emulsion PCR (emPCR) => 1 bead + 1 DNA fragment w/ linker (known sequ) per vesicle
  • OR surface PCR (solexa)
6
Q

Describe the 454 amplification method

A
  • emulsion PCR
  • DNA amplify on bead
  • each bead go into separate wells
  • pack wells w/ sequencing reagents: enzymes
  • pyrosequencing rxn
7
Q

Describe the sequencing helicos method

A
  • no amplification
  • dNTP labeled w/ dye
  • cycles of 1 dNTP per rxn -> image -> chemically cleave dye for next rxn cycle ->repeat
8
Q

Describe the sequencing pacific biosciences method

A
  • amplified DNA goes through pol. => sequenced
  • when base added = fluores
  • Hairpin loop inc accuracy
9
Q

Describe the sequencing illumina method

A
  • primers on slide = amplification

- labeled reversible terminator dNTP = detected by laser

10
Q

Describe the sequencing ion torrent method

A
  • detect pH change when dNTP is incorporated bc H+ released
11
Q

Describe next-next gen sequencing

A
  • DNA moves through nanopore => detected by “solid state”

- measure change in impedance (disruption in current)

12
Q

Research applications (5-7)

A
  • genome resequencing
  • targeted genome resequencing
  • cancer genomics
  • pharmacogenomics
  • transcriptome
  • methylation analysis
  • micro RNA profiling
13
Q

types of variations detected by next gen sequencing

A
  • point mutation
  • deletions & insertions
  • translocation breakpoint
  • pathogen
14
Q

challenges of clinical applications (Of next gen sequ)

A
  • methological challenges
  • changing from monogenic to polygenic disease
  • uncertainty of some results
  • predicted freq. of recessive mutations in pop