Scribd
Upload
10 views6 pages

L8 DNA Sequencing

Uploaded by

Maxwel Odongo
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
10 views6 pages

L8 DNA Sequencing

Uploaded by

Maxwel Odongo
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 6

1

Lecture 8: DNA Sequencing


Specific objectives
By the end of this lecture, you should be able to:
1 What is DNA sequencing
2 State the precise function of dideoxynucleotide in DNA sequencing.
3 State the structural difference between a ddNTP and dNTP.
4 Describe the following methods of DNA sequencing
(a) Dideoxy sequencing.
(b) Automated sequencing
5. Explain the general principles of DNA sequencing and describe DNA sequencing by the
dideoxy method.

Introduction
 DNA sequencing is the determination of the sequence of nucleotides making up a length
of DNA.
 The key to DNA sequencing involves starting with a defined fragment of DNA uniquely
labeled at one end, then generating a population of molecules that differ in size by one
base, and being able to separate these molecules and identify the base in each case.
 The technique involves placing single-stranded DNA on a special gel material
(acrylamide) and subjecting it to an electric current to separate strands on the basis of
their lengths.
 The mobility of a strand in inversely proportional to the logarithm of its length. This
technique is so sensitive that fragments differing in length by only a single nucleotide can
be separated.
 One of the requirements for DNA sequencing is the ability to obtain specific fragments of
DNA.
 There is a strong interdependence of DNA cloning and DNA sequencing technology,
since DNA cloning provides large amounts of specific DNA fragments.
Historical background
The first DNA molecule to be completely sequenced was the 5386 nucleotide genome of
bacteriophage ФX174, which was completed in 1973. This was followed by sequences for
SV40 virus (5243 bp) in 1977 and pBR322 (4363 bp) in 1978. Professor Sanger’s group
published the sequence of the human mitochondrial genome (16.6 kb) in 1981 and of
bacteriophage λ (49 kb) in 1982. Nowadays sequences of 10-20 kb are routine.
8.1 Dideoxy Sequencing
 This method of sequencing utilizes DNA polymerase to increase DNA chain length. It
has been termed the plus-minus method.
 It utilizes single-stranded DNA and dideoxy nucleotides, which lack a hydroxyl group.
The respective triphosphates can be incorporated into a growing chain, but they terminate
synthesis, because they do not permit bond formation with the next nucleotide
triphosphate.
 Four reaction tubes are each prepared with single-stranded DNA template for the
sequence of interest, plus DNA polymerase and a section of labeled primer.
 Each receives a small amount of a different dideoxynucleotide triphosphate (ddNTP),
together with the four normal deoxynucleotide triphosphates (dNTPs).
2

 In any given tube, various chain lengths will be produced, each corresponding to the
point at which the respective ddNTP for that tube was incorporated and terminated chain
length.
 These lengths, in turn, are an indication of where the bases complementary to the ddNTPs
occur on the template strand.
 The relative lengths can be visualized by running the four samples in four lanes on an
electrophoresis gel and the positions of the corresponding bases can be determined.
3
4

8.2 Procedure for sequencing DNA devised by Maxam and Gilbert

(a) The double-stranded DNA fragment to be sequenced is first labeled by attaching a


radioactive phosphorus (32P) group at the 5’ end of each strand.
(b) Dimethyl sulphoxide is then added and the labeled DNA sample heated to 90oC. This
results in breakdown of the base-pairing and dissociation of the DNA molecule into
its two component strands.
(c) The two strands are separated from one another by gel electrophoresis, which works
on the basis that one of the strands contains more purine nucleotides (adenine,
guanine) than the other and will therefore be slightly heavier.
(d) One strand is purified from the gel and divided into four samples, each of which is
treated with one of the cleavage reagents.
(e) The first set of reagents to be added cause a chemical modification in the nucleotides
they are specific for, making the strand susceptible to cleavage at that nucleotide
when an additional chemical, piperidine, is added. The modification and cleavage
reactions are carried out under conditions that result in only one cleavage per strand.
(f) Some of the cleavage fragments retain the 32P label at their 5’ ends.
(g) After electrophoresis, using the same special conditions as for chain termination
sequencing, the bands visualized by autoradiography will represent these labeled
fragments.
(h) The nucleotide sequence can now be read from the autoradiograph exactly as for
chain termination technique of sequencing.
5

8.3 Automated Sequencing


 The primer extension reactions are run in the same way as in the manual method, except
that the primers in each reaction are labelled with a different fluorescent molecule that
emits light of a distinct colour.
 The various primer extension reaction products separate according to size upon gel
electrophoresis.
 The bands are colour-coded according to the termination reaction that produced them e.g.
green for oligonucleotides ending in ddA, blue for those ending in ddC, and so forth.
 A laser scanner excites the fluorescent tag on each band as it passes by, and a detector
analyzes the colour of the resulting emitted light. This information is converted to a
sequence of bases and stored by a computer.
 The computer can produce a printout showing coloured peaks representing different
bases.
6

You might also like