Sequencing

Question 1

What is the importance of sequencing?

Answer 1

To know the sequence in a section of DNA

Question 2

What is a dideoxynucleotide (ddNTP)? & result of this

Answer 2

no -OH on 3’ C = stops elongation bc P-O bond can’t form w/out it

Question 3

How Sanger sequencing works

Answer 3

• 4x tube rxns: contain dNTP + ddNTP (1 type per tube)
• ssDNA template
• use radioactively labelled oligonucleotide primer
• each tube put in wells of gel electrophoresis=> read bottom-up to get sequence

Question 4

how Cycle sequencing works (aka automated sequencing)

Answer 4

• single tube rxn- requires [dNTP] = [ddNTP]
• ssDNA template
• fluorescent labels on ddNTP: diff. colour for each
• Use capillary array & laser to detect sequence

Question 5

how to interpret sequencing results

Answer 5

Read from smallest to longest strand (bottom-> up)

Question 6

List the diff. next generation sequencing & briefly explain its charact.

Answer 6

• Pyrosequencing: know which base incorporated due to l pyrophosphate released = ATP = light measured
• Illumina sequencing: sequencing by synhtesis
• Ion torrent sequencing: detect pH change when dNTP is incorporated bc H+ released
• Ion torrent (PGM) throughput: read DNA on site/field bc small device

Question 7

Uses/applications for next generation sequencing

Answer 7

• Transcriptome analysis, Gene regulation analysis
• mutation discovery
• RNA sequencing
• Whole genome gene expression
• Methylation analysis (for epigenetics)
• Forensics

Question 8

Distinguish between top-down vs bottom-up whole genome sequencing*

Answer 8

• Bottom-up: aka (whole-genome) shotgun sequencing. (Chromosome broken into) lots of large, random fragments (w/ RE) that overlap to cover genome
• Top-down: aka Hierarchal shot gun sequencing. A smaller scale than shotgun sequ. Long fragments of genomic DNA (100-300kb) obtained & cloned in Hi-capacity vector (BAC) -> cut in smaller pieces & cloned (subclones) -> sequence -> overlap & arrange sequence to see where it originated.
  This process is slow & expensive

Question 9

How are physical maps constructed?

Answer 9

by chromosome walking technique (similar to heirarchal shotgun sequ.): overlap clones to give order of the sequ.

Question 10

Automate sequencing means…

Answer 10

to acquire DNA sequence data in real time