9a - Detection of Genetic Variation 2 Flashcards
1
Q
DNA sequencing
A
Determining the nucleotide sequence of DNA
2
Q
Sanger technique / Dideoxy sequencing
A
- Uses dideoxynucleotides
- These are molecules that resemble normal nucleotides but lack the normal -OH group
3
Q
Dideoxy DNA sequencing steps 1-5
A
- DNA template is denatured to single strands.
- Single DNA primer (3’ end near sequence of interest) is annealed to template DNA and extended with DNA polymerase.
- Four reactions are set up
- A different labeled dideoxynucleotide is added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs
- ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal dNTPS, and produce no phosphodiester bond.
4
Q
Dideoxy DNA sequencing steps 7-10
A
- Whenever the labeled ddNTPs are incorporated in the chain, DNA synthesis terminates
- Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions.
- Extension products in each of the four reaction mixtures also end with a different labeled ddNTP (depending on the base).
- Gel electrophoresis is then conducted
5
Q
What do the four reactions of dideoxy DNA sequencing contain
A
- DNA template
- Primer annealed to template DNA
- DNA polymerase
- dNTPS
6
Q
Other name for dideoxy DNA sequencing
A
Dye terminator sequencing
7
Q
Polyacrylamide gel
A
Thinner–> higher voltage –> faster
8
Q
Pyrosequencing
A
- Sequencing by synthesis principle instead of chain termination with dideoxy nucleotides
- VERY expensive
9
Q
Steps of pyrosequencing
A
- Immobilise a single template DNA molecule on a bead/substrate and synthesise complementary strand
- Detect which nucleotide is added at each step, sequencing (polymerisation) doesn’t stop
- Complex reaction (template, primer, polymerase, extra enzymes)
- As with dideoxy sequencing, base incorporation is recorded when light is emitted at particular wavelengths
10
Q
Examples of NGS platforms
A
- Illumina
- 454/FLX
- Ion torrent PGM
11
Q
4 Steps of NGS
A
- Fragment target DNA and ligate universal adaptors
- Amplify single molecules (beads vs free)
- Sequence clonal amplicons
- Computer assembles data
12
Q
step three of NGS (sequence clonal amplicons)
A
- Each system differs in the way the DNA is sequenced
- Sequence 1 bp, read, repeat
13
Q
step four of NGS (computer assembles data)
A
- Reference based assemblies
- De novo assemblies
14
Q
Adapters
A
- Simplified sample processing
- Purification steps
- Uniform library quantification by qPCR
- Uniform amplification / sequencing
15
Q
Emulsion PCR
A
- All fragments have 2 adapters ligated
- Oil in water emulsion (1 bead and 1 DNA fragment per droplet)
- Clonal amplification on the bead
16
Q
Bridge PCR
A
- Prepare genomic DNA sample
- Attach DNA to surface
- Bridge amplification
- Fragments become double stranded
- Denature the ds molecules
- Complete amplification
- Determine first base
- Image first base
- Determine second base
- Image second chemistry cycle
- Sequence reads over multiple chemistry cycles
- Align data
17
Q
Characteristics of Ion Torrent
A
- Post light (no optics, fluorescence, or light)
- Rapid (no chemical reactions other than polymerase)
- Expandable (mostly solid-state)
- Measures changes in pH (due to release of H during replication)
18
Q
Nanopore sequencing
A
- One protein unzips the DNA helix into two strands
- A second protein creates a pore in the membrane and holds an “adaptor” molecule
- A flow of ions through the pore creates a current, each base blocks the flow to a different degree, altering the current
- The adaptor molecule keeps bases in place long enough for them to be read electronically
