DNA sequencing future developments

Question 1

5 phases of sequencing

Answer 1

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

Question 2

disadvantage to pyrosequencing

Answer 2

rxn vol inc = efficiency dec

*from adding nucleotides for each rxn cycle

Question 3

what techniques are combined in Next gen sequencing

Answer 3

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

Question 4

describe technique of next gen sequencing

Answer 4

• all use polymerase or ligase

- detect sequence using fluorescence or chemiluminescence

Question 5

Describe the general amplification technique in next gen

Answer 5

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

Question 6

Describe the 454 amplification method

Answer 6

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

Question 7

Describe the sequencing helicos method

Answer 7

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

Question 8

Describe the sequencing pacific biosciences method

Answer 8

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

Question 9

Describe the sequencing illumina method

Answer 9

• primers on slide = amplification

- labeled reversible terminator dNTP = detected by laser

Question 10

Describe the sequencing ion torrent method

Answer 10

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

Question 11

Describe next-next gen sequencing

Answer 11

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

- measure change in impedance (disruption in current)

Question 12

Research applications (5-7)

Answer 12

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

Question 13

types of variations detected by next gen sequencing

Answer 13

• point mutation
• deletions & insertions
• translocation breakpoint
• pathogen

Question 14

challenges of clinical applications (Of next gen sequ)

Answer 14

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