Sequencing Flashcards Preview

Techniques Course > Sequencing > Flashcards

Flashcards in Sequencing Deck (11):
0

Requirements for DNA replication

Template
Primer
dNTP bases
DNA polymerase

1

Sanger sequence overview

5' primer is radio labelled and annealed to template
DNTP and DNA polymerase added
Separated in 4 and ddNTps added
Gel electrophoresis
Detect radioactivity

2

How do ddNTps differ?

Lack 3'OH group needed to form phosphodiester bond with next nucleotide

3

How was Sanger sequence evolved.

DdNTps labeled with fluorochrome so can all happen in one tube
Standard gel electro replaced with capillary gel electro

4

Phred quality score

Measure of base call accuracy

5

Disadvantages if Sanger sequencing now

- not high throughput
- high cost per base

6

Contiguous sequences

- randomly fragment DNA
- clone fragments into plasmid
- sequence remove plasmid sequence
- assemble sequences into contiguous sequences

7

Basic principles of NGS

DNA fragmented
Ligate adaptors
Clonal amplification on solid surface
Primer, DNA polymerase, dNTPs
Sequencing by synthesis

8

How a SmRT cell works

- 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

How oxford nano pore works

- 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

Applications of NGS

- genome sequencing
- meta genome sequencing
- RNA sequencing
- amplicon or targeted sequencing
- ivf