9a - Detection of Genetic Variation 2

Question 1

DNA sequencing

Answer 1

Determining the nucleotide sequence of DNA

Question 2

Sanger technique / Dideoxy sequencing

Answer 2

• Uses dideoxynucleotides
• These are molecules that resemble normal nucleotides but lack the normal -OH group

Question 3

Dideoxy DNA sequencing steps 1-5

Answer 3

1. DNA template is denatured to single strands.
2. Single DNA primer (3’ end near sequence of interest) is annealed to template DNA and extended with DNA polymerase.
3. Four reactions are set up
4. A different labeled dideoxynucleotide is added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs
5. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal dNTPS, and produce no phosphodiester bond.

Question 4

Dideoxy DNA sequencing steps 7-10

Answer 4

7. Whenever the labeled ddNTPs are incorporated in the chain, DNA synthesis terminates
8. Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions.
9. Extension products in each of the four reaction mixtures also end with a different labeled ddNTP (depending on the base).
10. Gel electrophoresis is then conducted

Question 5

What do the four reactions of dideoxy DNA sequencing contain

Answer 5

1. DNA template
2. Primer annealed to template DNA
3. DNA polymerase
4. dNTPS

Question 6

Other name for dideoxy DNA sequencing

Answer 6

Dye terminator sequencing

Question 7

Polyacrylamide gel

Answer 7

Thinner–> higher voltage –> faster

Question 8

Pyrosequencing

Answer 8

• Sequencing by synthesis principle instead of chain termination with dideoxy nucleotides
• VERY expensive

Question 9

Steps of pyrosequencing

Answer 9

1. Immobilise a single template DNA molecule on a bead/substrate and synthesise complementary strand
2. Detect which nucleotide is added at each step, sequencing (polymerisation) doesn’t stop
3. Complex reaction (template, primer, polymerase, extra enzymes)
4. As with dideoxy sequencing, base incorporation is recorded when light is emitted at particular wavelengths

Question 10

Examples of NGS platforms

Answer 10

• Illumina
• 454/FLX
• Ion torrent PGM

Question 11

4 Steps of NGS

Answer 11

1. Fragment target DNA and ligate universal adaptors
2. Amplify single molecules (beads vs free)
3. Sequence clonal amplicons
4. Computer assembles data

Question 12

step three of NGS (sequence clonal amplicons)

Answer 12

• Each system differs in the way the DNA is sequenced
• Sequence 1 bp, read, repeat

Question 13

step four of NGS (computer assembles data)

Answer 13

• Reference based assemblies
• De novo assemblies

Question 14

Adapters

Answer 14

• Simplified sample processing
• Purification steps
• Uniform library quantification by qPCR
• Uniform amplification / sequencing

Question 15

Emulsion PCR

Answer 15

• All fragments have 2 adapters ligated
• Oil in water emulsion (1 bead and 1 DNA fragment per droplet)
• Clonal amplification on the bead

Question 16

Bridge PCR

Answer 16

1. Prepare genomic DNA sample
2. Attach DNA to surface
3. Bridge amplification
4. Fragments become double stranded
5. Denature the ds molecules
6. Complete amplification
7. Determine first base
8. Image first base
9. Determine second base
10. Image second chemistry cycle
11. Sequence reads over multiple chemistry cycles
12. Align data

Question 17

Characteristics of Ion Torrent

Answer 17

• 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)

Question 18

Nanopore sequencing

Answer 18

1. One protein unzips the DNA helix into two strands
2. A second protein creates a pore in the membrane and holds an “adaptor” molecule
3. A flow of ions through the pore creates a current, each base blocks the flow to a different degree, altering the current
4. The adaptor molecule keeps bases in place long enough for them to be read electronically