L4 & 5

Question 1

When did Sanger publish dideoxy sequencing

Answer 1

In 1977

Question 2

What does Sanger sequencing do

Answer 2

It relies on DNA polymerase to synthesize a
new strand of DNA, which in turn can reveal the sequence of the target DNA strand.

Question 3

What does DNA polymerase do

Answer 3

It replicates a new DNA strand complementary to a piece of single stranded DNA

Question 4

How does DNA pol replicate a new strand

Answer 4

by linking the 5ʹ-hydroxyl end of a free nucleotide to the 3ʹ-OH group of
the nucleotide at the end of a primer.

Question 5

What is a primer

Answer 5

a small piece of single-stranded DNA that can hybridize to one strand of the
template DNA and be extended by successive additions of nucleotides.

Question 6

Requirement of Sanger sequencing

Answer 6

a supply of nucleotide triphosphates (dNTPs) and 2ʹ,3ʹdideoxynucleotide triphosphates (ddNTPs) in small quantities relative to dNTPs

Question 7

ddNTPs contain no reactive 3ʹ-OH hence they are able to?

Answer 7

terminate DNA synthesis once they
are incorporated into the primer extension.

Question 8

How does DNA synthesis occur

Answer 8

in the 5’ to 3’ direction with the
new nucleotide being added
at the –OH terminus of the
3’carbon

Question 9

A typical reaction mixture contains

Answer 9

– DNA polymerase.
– DNA to be sequenced.
– DNA primers.
– (dNTPs) dATP, dTTP, dCTP, dGTG.
– Small quantity of one ddNTPs.
– Electrophoresis gel and rig.

Question 10

When does primer extension stop

Answer 10

until an unmatched nucleotide is paired with a complementary ddNTP.
Many fragments, each ending with a ddNTP of varying length, are produced from such a reaction

Question 11

What makes fragments detectable by radiography

Answer 11

Radioactive sulphur or phosphorus isotopes are incorporated into the newly synthesized DNA template via labelled dNTPs

Question 12

What is the goal of the human genome project

Answer 12

to identify all protein coding genes, sequence all chromosomes and make
available via biological publicly available databases.

Question 13

Key findings of the human genome project include

Answer 13

gene number (~22,000), alternative splicing,
high number of orthologs shared amongst vertebrates, human genome is rich in
transcription factors.

Question 14

Next Generation Sequencing

Answer 14

refers to novel, commercial technologies that allow us generate millions of sequence
reads in a single sequencing reaction. The length of each sequence read varies from 150-40,000 bp

Question 15

Roche 454 sequencing

Answer 15

is based on pyrosequencing (requires enzymes), a technique which detects
pyrophosphate release, using light signal (bioluminescence), after nucleotides are incorporated by
polymerase to a new strand of DNA.

Question 16

Illumina (Solexa) sequencing

Answer 16

works by simultaneously identifying DNA
bases, as each base emits a unique fluorescent signal, and adding them to a nucleic acid chain. Most commonly used

Question 17

Ion Torrent: Proton / PGM sequencing

Answer 17

g measures the direct release of H+
(protons) from the incorporation of individual bases by DNA polymerase.

Question 18

Steps of Roche 454 sequencing

Answer 18

1) Library preparation.
2) Clonal amplification.
3) Sequencing.

Question 19

Library preparation of Roche

Answer 19

DNA is fragmented into single stranded fragments (300-800 bp). Adaptors containing universal priming sites are ligated to target ends. This allows for fragment amplification with common PCR primers.

Question 20

Clonal amplification of Roche

Answer 20

DNA is mixed in with capture beads (~35µm). These contain adapter primers. Beads are added to PCR reagents and synthetic oil to produce an emulsion.

Question 21

Steps of Illumina Genome Analyser

Answer 21

1) Library preparation.
2) Clonal clusters amplification.
3) Sequencing.

Question 22

Library preparation of illumina

Answer 22

DNA fragmented by neubilization or sonication and sequence adapters
are added

Question 23

Where does cluster amplification occur

Answer 23

On a flow cell

Question 24

What is a flow cell

Answer 24

A thick glass slide with channels or lanes. Each lane is coated with a lawn of oligos that are complementary to library adapters

Question 25

Sequencing of illumina

Answer 25

*Flow cell transferred to IGA and sequencing primers, polymerases and
deoxynucleoside triphosphates (dNTP) are added.

Question 26

Limitation of second generation sequencers

Answer 26

short read lengths (300bp)

Question 27

What are third generation sequencers

Answer 27

those capable of sequencing single
molecules without the need for DNA amplification

Question 28

PacBio sequencing or SMRT sequencing

Answer 28

offers much longer read lengths and faster runs than second generation methods but is hindered by a lower throughput, higher
error rate, and higher cost per base.