11.3 DNA Sequencing

Question 1

Sanger dideoxy chain terminating method

Answer 1

Four tubes, one for each base and containing 5% dd_TPfor the specific nucleotide. Incorporation of the dd_TP leads to termination of chains producing many fragments of different sizes that correspond with the locations of certain bases. Gel electrophoresis then reveals the address of each base.

Question 2

Fluorescent dideoxy sequencing

Answer 2

Uses denaturing polyacrylamide gel and as ddNTP fragments migrate through the gel they pass a laser beam and fluorescent dyes (specific for each) are excited.

Question 3

Massively parallel sequencing

Answer 3

Illuminati
Torrent
- Sequencing by synthesis

Question 4

Illumina sequencing key points

Answer 4

Short DNA segments are sheared and adaptor sequences are lighted to the ends to add sites for primers and enable attachment to the oligonucleotides.
Uses bridge amplification.

Question 5

Illumina sequencing process

Answer 5

Addition of fluorescently labelled nucleotide analogs that are reversible chain terminators. Pause after each dNTP addition - laser photo - continue for hundreds of cycles.
Millions of distinct DNA sequences are determined simultaneously.

Question 6

Nanopore sequencing

Answer 6

Single molecule at a time, enzyme unwinds DNA so it can be pulled through a pore using an electrical current. Each base causes a unique disturbance in the current. Less accurate.