L6 Next gen sequencing

Question 1

First generation

Answer 1

Dideoxynucleotide termination,

electrophoresis,

Sanger method

Question 2

Second generation

Answer 2

Sequencing by synthesis,

wash and detect approach,

454 Pyrosequencing (roche),

illumina

Question 3

Third generation (most important)

Answer 3

Single molecule sequencing,

true single molecule sequencing (Helicos),

Nanopore sequencing (oxford nanopore technologies).

This does not require the dna amplification step!!

Question 4

454 sequencing (sequencing)

Answer 4

Uses 4 enzymes acting in sequence, DNA Pol > Ppi produce, reacts with adenosine 5 phosphate to produce ATP > ATP + Lucerferin > Light. 300-800 base pairs fragments. DNA denatured into single strands, which are then binded to microbeads, there are enzymes which will multiply the DNA many times. 2 million copies of the same single strand DNA on each bead. The beads are then put on picotitre plate (1.6 million wells) the wells hold a single bead. When nucleotides are added, light will be produced.

Question 5

illumina sequencing

Answer 5

1) Fragmentation 2) Bridge amplification which forms clusters containing single strands 3) nucleotides which are added have flouresent groups and a blocking group so only 1 can be added at a time. 4) once the nucleotide is added the blocking group is removed and the flouresence is given out. next nucleotide is added.

Question 6

What is the difference between Sanger, Pyrosequencing, Illumina, SOLiD

Answer 6

Sanger - 800bp 96kb per run

Question 7

Single DNA molecule sequencing (Helicos)

Answer 7

Template DNA is modified using termina transferase, which adds polyA, the last A carries a flouresent group. This is attactched to a complimentary surface. each nucleotide added carries a flouresent group. When they are added, flouresence is given out. the excess nucleotide is washed away, the flouresence group is enzymaticaly removed, then it is repeated.

Question 8

Nanopore sequencing

Answer 8

A synthesised nanopore is made. A molecule of any kind is passed through the nanopore under the influence of an electric field. The deviation under the field is then recorded. The nanopore is then modified to allow 1 nucleotide to pass via cyclodextrin. Each nucleotide has a different size and shape hence, they can be differentiated once they pass through the current. This does not require DNA amplification.

Question 9

Reading material

Answer 9

user = swansea pass = member Henry stewart talks