Week 8 - Sequencing

Question 1

Name the 2 original methods for DNA sequencing

Answer 1

Maxam and Gilbert (Chemical cleavage)
Sanger and Coulson (Chain termination)

Question 2

Next generation sequencing (NGS)

Answer 2

Pyrosequencing
Ligation Sequencing
SMRT (Single molecule Real time) sequencing
Ion semiconductor sequencing (MINION)
Reversible terminator sequencing (Illumina)

Question 3

Sanger method

Answer 3

controlled inhibition of enzymatic DNA synthesis, uses dideoxy nucleotides to terminate polymerization, these lack an OH group but are processed like normal nucleotides.

Question 4

Requirements needed for the Chain termination sequencing

Answer 4

multiple copies of ss template DNA
primers
DNA polymerase
dNTPS
small amount of radioactively-labelled dideoxynucleotides (ddNTPs)

Question 5

What is denaturing polyacrylamide gel used for?

Answer 5

Used in combination with the chain termination method, to determine DNA products by base e.g. = A,T,C,G

Question 6

What is the problem with the chain termination method and what is done to solve it?

Answer 6

chain termination only sequences 500-800 bases, many genes are longer than this.

The 2 approaches used are Fragmentation and walking

Question 7

What is the fragmentation method used with chain termination?

Answer 7

It involves cutting the DNA insert into fragments before sequencing them and them aligning and overlapping the reads in a consensus sequence.

Question 8

What is sequence walking?

Answer 8

it is a method used with chain termination, as CT cannot exceed 800 bases to obtain a full sequence you must walk down the sequence. Requires design of successive primers based the previously obtained sequences.

Question 9

Pros and Cons of Chain termination sequencing

Answer 9

Pros - accurate sequencing of 800bp and checking recombinant constructs
cons - slow, not scalable, expensive per base sequenced,