What is the importance of sequencing?
To know the sequence in a section of DNA
What is a dideoxynucleotide (ddNTP)? & result of this
no -OH on 3’ C = stops elongation bc P-O bond can’t form w/out it
How Sanger sequencing works
- 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
how Cycle sequencing works (aka automated sequencing)
- single tube rxn- requires [dNTP] = [ddNTP]
- ssDNA template
- fluorescent labels on ddNTP: diff. colour for each
- Use capillary array & laser to detect sequence
how to interpret sequencing results
Read from smallest to longest strand (bottom-> up)
List the diff. next generation sequencing & briefly explain its charact.
- 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
Uses/applications for next generation sequencing
- Transcriptome analysis, Gene regulation analysis
- mutation discovery
- RNA sequencing
- Whole genome gene expression
- Methylation analysis (for epigenetics)
- Forensics
Distinguish between top-down vs bottom-up whole genome sequencing*
- 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
How are physical maps constructed?
by chromosome walking technique (similar to heirarchal shotgun sequ.): overlap clones to give order of the sequ.
Automate sequencing means…
to acquire DNA sequence data in real time
