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GENETICS gts251
A Conceptual Approach
Benjamin A Pierce 7th Ed (Digital Update)

CHAPTER 19
Molecular Genetic Analysis and Biotechnology

LECTURE 6.5
p 582 – 588
DNA sequencing
Reminder from introduction lecture
What is expected of you in GTS 251?
• Take administrative responsibility.
• Prepare the specified material as outlined in the semester planner.
Read the indicated pages before class.
• Attend lectures.
• Do tutorial assignments and online Achieve activities.
• Attend tutorials.
• Make optimal use of lectures and tutorial sessions.
• Think and ask questions.
• Involve yourself with the course content on a continuous basis. Use the
study guide as an aid to study from the textbook. Test yourself at the
end of a study unit by using the learning outcomes and self-study
questions in the study guide.
• Take academic responsibility.
Principles for SU 6 & 7:

• You are expected to prepare and work through the indicated

material from the text book prior to each lecture.
• Slides are designed to be used as study aid in conjunction
with the text book, and represent concepts which I want to
mention in class. Slides are NOT meant to serve as sole
source of material for content mastering or study.
• Lecture time will be used to revise important concepts,
highlight misconceptions, and interactively work through
examples and problems.
• Post-lecture notes containing class annotations will be posted.
 LECTURE 6.5
LEARNING OBJECTIVES
At the end of this lecture, you should be able to describe and apply
molecular techniques related to DNA sequencing.

• List the components of a Sanger sequencing reaction, and provide

a function for each.
• Describe how the base sequence of a DNA fragment is determined.
• Use a DNA sequence to predict the sequencing gel profile, or use a
gel profile to determine the sequence of the new strand or the
template strand.
• Explain the differences in the principles underlying Sanger (dideoxy)
DNA sequencing and next-generation and third-generation
sequencing technologies.
 19.4 DNA Sequences Can Be Determined and Analyzed

Dideoxy Sequencing

• DNA sequencing is the process whereby the sequences of bases in

DNA is determined.
• Sanger dideoxy sequencing is based on replication, which is
sometimes terminated when a specific base is encountered.
• A dideoxyribonucleoside triphosphate (ddNTP) lacking a 3′-OH
group is used, which terminates DNA synthesis when incorporated.
 Modified
Normal ddNTP
dNTP

no 3’OH available,
so chain termination
Sanger sequencing reaction

• Solution containing multiple copies of purified template DNA

(denatured or ssDNA) is split into four tubes.
• To each tube add:
– a primer,
– all four normal dNTPs,
– one ddNTP (either ddATP, or ddCTP, or ddGTP, or ddTTP),
– DNA polymerase.
• Primer or one of the dNTPs must be radioactively or chemically
labeled.
What happens in the tube?

• The primer binds to its complementary sequence on the template.

• DNA polymerase extends it from the 3’OH, and synthesises a new
strand complementary to the template.
• Usually normal dNTPs are used for synthesis, occasionally a ddNTP
is incorporated into the new strand.
• Incorporation of a ddNTP teminates replication of that specific strand.
• In e.g. the tube containing ddATP, a set of fragments of different
lengths will be produced that were all terminated opposite a T in the
template.
• Equivalent reactions take place in the other 3 tubes, terminating
opposite A, C or G.
• Contents of four tubes are separated side by side on an acrylamide
gel.
• Samples are visualised, e.g. by autoradiography.
• DNA sequence of the newly synthesised strand is read from the gel.
• This is the complement of the original template strand sequence.

NB: Also view the animation and problem solving video

Achieve – SU 6 Ch 19 – Resources – Interactive Video Assignment:
Dideoxy Sequencing of DNA.
Achieve – SU 6 Ch 19 – Resources – Problem Solving Video:
Sanger (Dideoxy) Sequencing.
PS:
You have the following ssDNA template available, with a primer annealed
as indicated. You add DNA polymerase, all dNTPs and ddTTP to the
tube, and incubate it at an appropriate temperature.

Give the sequence of any of the new strands (5’ – 3’) which will be
synthesized in this tube. Type bases only, without spaces or polarity.
Rank Responses
1 GAT
2 GATT
3 GATTCGAGCTGA
4 GA
5 GATTCGAGCT
6 GATTCGA
Dideoxy Sequencing of DNA

View Transcript
Example question:
You want to sequence the following DNA fragment using the Sanger
dideoxy sequencing method. Draw the bands that should appear on the
gel from the four sequencing reactions.

5’ TCGGATCAA-primer site 3’
PS:
You obtain the following sequencing gel image. Give the base
sequence of the newly synthesized DNA strand.

A. 5’ CATGATCGT 3’
B. 5’ ACGATCATG 3’
C. 5’ TGCTAGTAC 3’
D. 5’ GTACTAGCA 3’
PS:
You obtain the following sequencing gel image. Give the base
sequence (5’ – 3’) of the original fragment.
Type answer, give bases only without spaces or polarity eg ATT…

Rank Responses
1 ATGACTACG
2 GCATCAGTA
3 TACTGATGC
4 CGTAGTCAT
5 ATGACACG
6 ATGATACG
Automation of Sanger sequencing:

• The ddNTPs are labeled with fluorescent dyes, a different colour dye
is used for each of the four ddNTPs.
• The four termination reactions take place in the same tube.
• The fragments are separated in gel-containing capillary tubes.
• A laser beam and detector record the fluorescence emitted by each
fragment.
• The results are peaks of different colour on a graph.
• The sequence information is read directly into a computer.
Terminators are colour-labelled, and all four reactions take place in the
same tube.

3’
C
A
T
G
A
T
C
G
T
5’

All fragments are separated by

Instead of a gel with 4 electrophoresis in one capillary tube,
different lanes and distinguished by colour detected
by a laser
 Next-Generation and Third-Generation
Sequencing Technologies

• New technologies have revolutionised sequencing applications.

• Millions of fragments can be sequenced simultaneously.
• Cheap.
Illumina sequencing

• Currently the most widely used sequencing technology.

• Similar in principle to Sanger dideoxy method.
• Special nucleotides with fluorescent tags and terminator groups are
utilised.
• Multiple fragments are sequenced simultaneously in flow cells.
• Fluorescence is monitored and recorded per cell as each new
nucleotide is added to a new DNA strand complementary to the
template.
Illumina:
Primers, DNA pol and
special dNTPs are added
to flow cell.
Primer anneals to
template, DNA synthesis
starts.

When T is incorporated, green

signal is recorded for that cell.
Thousands of cells with different
templates recorded in parallel.
Tag and terminator are removed.
New dNTPs and
polymerase added.

When G is incorporated, blue signal is

recorded for that cell.
Cycle repeats.
Sequence is read as a series of flashes of
coloured light from each cell.

From this cell TGGCATGTC ….. will be

recorded.
Third-generation sequencing technologies

• More advanced third-generation sequencing technologies which are

also currently being used can determine the sequence of single
molecules of DNA or RNA.
• Allow much longer fragments to be sequenced.
• These include PacBio and Oxford nanopore sequencing.
ARE YOU NOW ABLE TO?

• List the components of a Sanger sequencing reaction, and provide

a function for each.
• Describe how the base sequence of a DNA fragment is determined.
• Use a DNA sequence to predict the sequencing gel profile, or use a
gel profile to determine the sequence of the new strand or the
template strand.
• Explain the differences in the principles underlying Sanger (dideoxy)
DNA sequencing and next-generation sequencing technologies.