Sequencing Flashcards
0
Q
Sanger sequence overview
A
5' primer is radio labelled and annealed to template DNTP and DNA polymerase added Separated in 4 and ddNTps added Gel electrophoresis Detect radioactivity
1
Q
Requirements for DNA replication
A
Template
Primer
dNTP bases
DNA polymerase
2
Q
How do ddNTps differ?
A
Lack 3’OH group needed to form phosphodiester bond with next nucleotide
3
Q
How was Sanger sequence evolved.
A
DdNTps labeled with fluorochrome so can all happen in one tube
Standard gel electro replaced with capillary gel electro
4
Q
Phred quality score
A
Measure of base call accuracy
5
Q
Disadvantages if Sanger sequencing now
A
- not high throughput
- high cost per base
6
Q
Contiguous sequences
A
- randomly fragment DNA
- clone fragments into plasmid
- sequence remove plasmid sequence
- assemble sequences into contiguous sequences
7
Q
Basic principles of NGS
A
DNA fragmented Ligate adaptors Clonal amplification on solid surface Primer, DNA polymerase, dNTPs Sequencing by synthesis
8
Q
How a SmRT cell works
A
- each cell patterned with zero mode wavelengths (ZMWs) and one DNA polymerase
- nucleotides diffuse into chamber
- each tagged with fluorescent marker
- only nucleotides near bottom fluoresce
- light-sensitive cameras collect pulses
- converted into base call
9
Q
How oxford nano pore works
A
- one protein unzips DNA helix
- second protein creates a pore in the membrane
- flow if ions creates a current
- adaptor molecule keeps bases in place long enough for them to be identified
10
Q
Applications of NGS
A
- genome sequencing
- meta genome sequencing
- RNA sequencing
- amplicon or targeted sequencing
- ivf
