INTRODUCTION TO

GENETIC ENGINEERING

BTG 345
A first semester course for 300l students of the Department of
Biological Sciences

Course Instructor I: Prof. George Ude (Ph.D.)

Course Instructor II: Michael Okoro (M.Sc.)
 STUDY OUTLINE
 INTRODUCTION TO BIOTECHNOLOGY
 Definition, Timeline, current uses
 Biotechnology tools & practical applications
 INTRODUCTION TO GENETIC ENGINEERING
 Steps in Gene Cloning Experiment
 Application of Genetic Engineering
 BRIEF OVERVIEW OF THE MOLECULAR BIOLOGY OF THE CELL
 The way that living systems are organized
 The flow of genetic information
 The structure of DNA and RNA
 Gene organization
 WORKING WITH NUCLEIC ACIDS
 Laboratory requirements
 Isolation of DNA and RNA
 Handling and quantification of nucleic acids
 Labelling nucleic acids
 Nucleic acid hybridization
 Gel electrophoresis
 DNA sequencing
 What is Biotechnology?
"Biotechnology" means any
technological application that uses
biological systems, living organisms, or
derivatives thereof, to make or modify
products or processes for specific use.

Definition from Convention on Biological Diversity

https://www.cbd.int/
3
 Biotechnology Over Time
Traditional Biotechnology
• Growing plants
• Raising animals
• Plant and animal breeding
• Fermentation (bread, beer, wine, fish sauce)

Genetic Engineering - Recombinant DNA and

tissue-culture-based biotechnology
• Genome Editing – Precision breeding

4
Current Uses of Biotechnology
Agriculture
• Transgenic Plants [disease resistance, drought
tolerance, nutrient use efficiency, plant-based
products such as vaccines]
• Transgenic Animals
• Transgenic Microbes
Pharmaceutical
• Insulin
• Antibiotics
• Cancer therapy
Others
• Mining
• Petroleum spill clean-up with microbes
5
Advanced Biotechnology Definition

is the use of biological systems,

such as microorganisms, whole
cells or their molecules, to solve
problems or to make useful
products.
Tools of Biotechnology
1. Bioprocessing technology
• Using whole living cells or components of them
to manufacture desired products.
• Most common whole cells used are yeast and
bacteria (one-celled organisms).
• Most common components are enzymes (proteins
that catalyze chemical reactions).
• Microbial fermentation
• Cells isolated from animals and plants also are
used to produce desired products.
Tools of Biotechnology
2. Genetic engineering
• The technique of removing, modifying or adding genes
to a DNA molecule to change the information it
contains.

• Is known more specifically as recombinant DNA

(rDNA) technology.

• The product of rDNA technology is known as a

genetically modified organism, or GMO.

• Example: Gene for human insulin inserted into E.coli

→ bacteria that make human insulin (a
biopharmaceutical).
Practical Applications of
Biotechnology
1. Agricultural applications
 Better crops
 Improved animal health

2. Medical and health care applications

 New tests
 New vaccines
 New medicines

3. Chemical and environmental applications

 Better manufacturing processes
 Improved consumer products
WHAT IS GENETIC ENGINEERING?
 Genetic engineering is the direct (deliberate)
manipulation of an organism’s gene using
biotechnology. It is a set of technologies used to change
the genetic makeup of cells, including the transfer of
genes within and across species boundaries to produce
improved or novel organisms.

 Other terms that can be used to describe the technology,

including gene manipulation, gene cloning,
recombinant DNA technology, genetic modification,
and the new genetics.
INTRODUCTION TO GENETIC ENGINEERING

Figure 1: Concept map 1

STEPS IN GENE CLONING EXPERIMENT
Although there are many diverse and complex techniques involved, the basic principles of genetic
manipulation are reasonably simple. The premise on which the technology is based is that genetic
information, encoded by DNA and arranged in the form of genes, is a resource that can be manipulated in
various ways to achieve certain goals in both pure and applied science and medicine.

Figure 2: The four steps in a gene cloning experiment. The term ‘clone’ comes from the colonies of
identical host cells produced during amplification of the cloned fragments. Gene cloning is sometimes
referred to as ‘molecular cloning’ to distinguish the process from the cloning of whole organisms.
STEPS IN GENE CLONING EXPERIMENT

Figure 3: Cloning DNA fragments. (a) The source DNA is isolated and fragmented into suitably
sized pieces. (b) The fragments are then joined to a carrier molecule or vector to produce
recombinant DNA molecules. In this case, a plasmid vector is shown. (c) The recombinant DNA
molecules are then introduced into a host cell (a bacterial cell in this example) for propagation as
clones.
APPLICATIONS OF GENETIC ENGINEERING
There are many areas in which genetic manipulation is
of value, including the following:

 Basic research on gene structure and function

 Production of useful proteins by novel methods
 Generation of transgenic plants and animals
 Medical diagnosis and treatment
 Genome analysis by DNA sequencing
 BRIEF OVERVIEW OF THE MOLECULAR
BIOLOGY OF THE CELL

 Living systems are organised hierarchically, with close

interdependence of structure and function.

 Two premises are useful here:

 First, there is a very close link between structure and function in
biological systems.

 Second, living systems provide an excellent example of the

concept of emergent properties.

 Cell is the basic unit of organisation in biological

systems; prokaryotic cells have no nucleus, eukaryotic
cells do
BRIEF OVERVIEW OF THE MOLECULAR
BIOLOGY OF THE CELL

Figure 4: Concept map 2

BRIEF OVERVIEW OF THE MOLECULAR
BIOLOGY OF THE CELL CONTD.

Figure 5 & 6: Cross-Section of an Animal Cell and Plant Cell

Source: EnchantedLearning.com
 BRIEF OVERVIEW OF THE MOLECULAR
BIOLOGY OF THE CELL CONTD.

 The chemistry of living systems is based on the

element carbon, which can form four covalent bonds
with other atoms. By joining carbon atoms together,
and incorporating other atoms, molecules can be built
up, which in turn can be joined together to produce
macromolecules.

 Biologists usually recognise four groups of

macromolecules: lipids, carbohydrates, proteins,
and nucleic acids.
 BRIEF OVERVIEW OF THE MOLECULAR
BIOLOGY OF THE CELL CONTD.
 The synthesis of macromolecules involves a condensation
reaction between functional groups on the molecules to be
joined together. This dehydration synthesis forms a
covalent bond by removing the elements of water.

 polymers can be broken apart into their constituent

monomers by adding the elements of water back to
reconstitute the original groups. This is known as
hydrolysis (literally hydro lysis, water breaking). The
monomer/polymer cycle and dehydration/hydrolysis are
illustrated in Figure 7.
 BRIEF OVERVIEW OF THE MOLECULAR
BIOLOGY OF THE CELL CONTD.

Figure 7: The monomer–polymer cycle. In this example a representation of amino acids and proteins is shown.
The amino acids (monomers) are joined together by removal of the elements of water (H2O) during dehydration
synthesis. When the protein is no longer required, it may be degraded by adding back the H2O during hydrolysis.
Although the cycle looks simple when presented like this, the synthesis of proteins requires many components
whose functions are coordinated during the complex process of translation.
BRIEF OVERVIEW OF THE STRUCTURE
AND FUNCTION OF NUCLEIC ACIDS
THE FLOW OF GENETIC INFORMATION
 Life is directed by four nitrogenous bases: adenine (A), guanine (G),
cytosine (C), and thymine (T). So how do these bases enable cells to
function?

 The expression of genetic information is achieved ultimately via proteins,

particularly the enzymes that catalyse the reactions of metabolism. Proteins
are condensation heteropolymers synthesised from amino acids, of which
20 are used in natural proteins.

 The flow of genetic information is unidirectional, from DNA to protein, with

messenger RNA (mRNA) as an intermediate. The copying of DNA-encoded
genetic information into RNA is known as transcription (TC), with the
further conversion into protein being termed translation (TL). This concept
of information flow is known as the Central Dogma of molecular biology
and is an underlying theme in all studies of gene expression.
 BRIEF OVERVIEW OF THE STRUCTURE
AND FUNCTION OF NUCLEIC ACIDS
 Duplication of the genetic material prior to cell division represents a DNA--
DNA transfer, known as DNA replication.

 Some viruses have RNA instead of DNA as their genetic material. These
viruses (chiefly members of the retrovirus group) have an enzyme called
reverse transcriptase (an RNA-dependent DNA polymerase) that produces
a double-stranded DNA molecule from the single-stranded RNA genome.

Figure 8: Central Dogma of life

BRIEF OVERVIEW OF THE STRUCTURE AND
FUNCTION OF NUCLEIC ACIDS
Table 1: The genetic code

Note: Codons read 5→3; thus, AUG specifies Met. The three-letter abbreviations for the amino acids are as
follows: Ala, Alanine; Arg, Arginine; Asn, Asparagine; Asp, Aspartic acid; Cys, Cysteine; Gln, Glutamine; Glu,
Glutamic acid; Gly, Glycine; His, Histidine; Ile, Isoleucine; Leu, Leucine; Lys, Lysine; Met, Methionine; Phe,
Phenylalanine; Pro, Proline; Ser, Serine; Thr, Threonine; Trp, Tryptophan; Tyr, Tyrosine; Val, Valine. The three
codons UAA, UAG, and UGA specify no amino acid and terminate translation.
 THE STRUCTURE OF DNA AND RNA
 In most organisms, the primary genetic material is double-stranded
DNA.

 Nucleic acids are polymers composed of nucleotides; DNA is

deoxyribonucleic acid, RNA is ribonucleic acid.

 The two types of nucleic acid (DNA and RNA) are named
according to the sugar component of the nucleotide, with DNA
having 2-deoxyribose as the sugar (hence DeoxyriboNucleicAcid)
and RNA having ribose (hence RiboNucleicAcid).

 Nucleic acids are heteropolymers composed of monomers known as

nucleotides; a nucleic acid chain is therefore often called a
polynucleotide. The monomers are themselves made up of three
components: a sugar, a phosphate group, and a nitrogenous base.
 THE STRUCTURE OF A NUCLEOTIDE

Figure 9: The structure of a nucleotide. Carbon atoms are represented by solid circles, numbered 1to 5. In
DNA the sugar is deoxyribose, with a hydrogen atom at position X. In RNA the sugar is ribose, which has
a hydroxyl group at position X. The base can be A, G, C, or T in DNA, and A, G, C, or U in RNA.
BASE PAIRING ARRANGEMENT IN DNA

Figure 10: Base-pairing arrangements in DNA. (a) An A·T base pair. The bases are
linked by two hydrogen bonds (dotted lines). (b) AG·C base pair, with three hydrogen
bonds.
DOUBLE HELIX STRUCTURE OF DNA

 Watson and Crick putting together

the physical and chemical evidence
with the X-defraction diagram
proposed a ‘double-stranded helix’
for the DNA structure.
 Sugar-phosphate
backbone form a helical
outer structure
 The bases are in the
center between two
chains

Figure 11: The double helix. This is DNA in the commonly found B-form. The right-handed helix
has a diameter of 2 nm and a pitch of 3.4 nm, with 10 base pairs per turn. The sugar–phosphate
‘backbones’ are antiparallel (arrowed) with respect to their 5→3 orientations. One of the sugar–
phosphate chains has been shaded for clarity. The purine-pyrimidine base pairs are formed across
the axis of the helix.
 THE DNA MOLECULE

Watson and Crick – 1953

• Double helical structure
•Polynucleotide chain
•Nucleotides – base, sugar
phosphate
• Adenine, Thymine, Cytosine
Guanine
•A/T; C/G
Figure 12: DNA Molecule
Source: http://www.ehrig-privat.de/ueg/images/dna-structure.jpg
 STRUCTURE OF RNA
 The structure of RNA is similar to that of DNA; the main chemical differences are
the presence of ribose instead of 2-deoxyribose and uracil instead of thymine. RNA
is also most commonly single stranded, although short stretches of double-stranded
RNA may be found in self-complementary regions.

 There are three main types of RNA molecule found in cells: messenger RNA
(mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

 Ribosomal RNA is the most abundant class of RNA molecule, making up some 85%
of total cellular RNA. It is associated with ribosomes, which are an essential part of
the translational machinery.

 Transfer RNAs make up about 10% of total RNA and provide the essential
specificity that enables the insertion of the correct amino acid into the protein that is
being synthesised.

 Messenger RNA, as the name suggests, acts as the carrier of genetic information
from the DNA to the translational machinery and usually makes up less than 5% of
total cellular RNA.
 GENE ORGANISATION
 The gene is the basic unit of genetic information. Gene encode information for a
particular protein (or RNA molecule)
 Genes are located on chromosomes at a particular genetic locus. Different forms of
the same gene are known as alleles.
 Genes have several important regions. A promoter is necessary for RNA polymerase
binding, with the transcription start and stop sites defining the transcriptional unit.

Figure 13: Gene organisation. The transcriptional unit produces the RNA molecule and is defined by the
transcription start site (TC) and stop site (tC). Within the transcriptional unit lies the coding sequence, from
the translation start site (TL) to the stop site (tL). The upstream regulatory region may have controlling
elements such as enhancers or operators in addition to the promoter (P), which is the RNA polymerase
binding site.
 GENE STRUCTURE IN PROKARYOTES
 In prokaryotic cells such as bacteria, genes are usually found grouped
together in operons.
 The operon is a cluster of genes that are related (often coding for enzymes in
a metabolic pathway) and that are under the control of a single
promoter/regulatory region.
 Perhaps the best known example of this arrangement is the lac operon,
which codes for the enzymes responsible for lactose catabolism.

Figure 14: The lac operon. The structural genes lacZ, lacY, and lacA (noted as z, y, and a) encode ß-galactosidase,
galactoside permease, and a transacetylase, respectively. The cluster is controlled by a promoter (P) and an operator
region (O). The operator is the binding site for the repressor protein, encoded by the lacI gene (i). The repressor
gene lies outside the operon itself and is controlled by its own promoter, Pi.
 GENE STRUCTURE IN EUKARYOTE
 A major defining feature of eukaryotic cells is the presence of a membrane-bound
nucleus, within which the DNA is stored in the form of chromosomes.

 Eukaryotic genes tend to be more complex than prokaryotic genes and often contain
intervening sequences (introns). The introns form part of the primary transcript,
which is converted to the mature mRNA by RNA processing.

Table 2: Size and structure of some human genes

 GENE EXPRESSION

Figure 15: Structure and expression of the mammalian ß-globin gene. The gene contains
two intervening sequences or introns. The expressed sequences (exons) are shaded and
numbered. The primary transcript is processed by capping, polyadenylation, and splicing
to yield the fully functional mRNA.
 GENE EXPRESSION - Transcription and
Translation
 These two processes are the critical steps involved in producing
functional proteins in the cell. Transcription involves synthesis of
an RNA from the DNA template provided by the non-coding strand
of the transcriptional unit in question. The enzyme responsible is
RNA polymerase (DNA-dependent RNA polymerase).

 In prokaryotes there is a single RNA polymerase enzyme, but in

eukaryotes there are three types of RNA polymerase (I, II, and
III).

 Transcription has several component stages: (1)DNA/RNA

polymerase binding, (2)chain initiation, (3)chain elongation, and
(4)chain termination and release of the RNA
 GENE EXPRESSION - Transcription and
Translation
 Translation requires an mRNA molecule, a supply of charged tRNAs
(tRNA molecules with their associated amino acid residues), and ribosomes
(composed of rRNA and ribosomal proteins).

 The ribosomes are the sites where protein synthesis occurs.

 The codon/anticodon recognition event marks the link between nucleic acid
and protein.

 Prokaryotic genes are often regulated in response to external signals such

as nutrient availability.

 Eukaryotic genes are often regulated in response to signals generated from

within the organism.
GENE EXPRESSION - Transcription and
Translation

Figure 16: Transcription and

translation. (a) Transcription involves
synthesis of mRNA by RNA
polymerase. (b) The ribosome is the
site of translation and is made up of the
large subunit (LSU) and the small
subunit (SSU). There are three sites
within the ribosome. The A
(aminoacyl) and P (peptidyl) sites are
involved in insertion of the correct
tRNA–amino acid complex in the
growing polypeptide chain. The E
(exit) site facilitates the release of the
tRNA after peptide bond formation has
removed its amino acid. (c) The mRNA
is being translated. The amino acid
residue is inserted into the protein in
response to the codon/anticodon
recognition event in the ribosome. The
ribosome translates the mRNA in a
5→3 direction, with the polypeptide
growing from its N terminus. The
residues in the polypeptide chain are
joined together by peptide bonds.
 GENES AND GENOMES
Table 3: Genome size in some organisms
 GENOME SIZE AND COMPLEXITY
 The amount of DNA in the haploid genome is known as the
C-value.

 Eukaryotic genomes may have a range of different types of

repetitive sequences.

 Most of the human genome is not involved in coding for

proteins. only about 3% of the total amount of DNA is
actually coding sequence. Even when the introns and control
sequences are added, the majority of the DNA has no
obvious function. This is sometimes termed ‘junk’ DNA,
GENOME SIZE AND COMPLEXITY CONTD.
 Genome sequencing has greatly improved our
understanding of how genomes work.

 Analysis of the transcriptome and proteome provides

useful information about which genes a cell is expressing
at any given time
WORKING WITH NUCLEIC ACIDS

Figure 17: Concept map 3

 WORKING WITH NUCLEIC ACIDS
 Every gene manipulation experiment requires a source of
nucleic acid, in the form of either DNA or RNA.

 It is therefore important that reliable methods are available for

isolating these components from cells. There are three basic
requirements:
 (1) opening the cells in the sample to expose the nucleic acids for
further processing,
 (2) separation of the nucleic acids from other cell components, and
 (3) recovery of the nucleic acid in purified form. A variety of
techniques may be used, ranging from simple procedures with few
steps up to more complex purifications involving several different
stages.
WORKING WITH NUCLEIC ACIDS
Figure 18: Preparation of
mRNA by affinity
chromatography using
oligo(dT)-cellulose. (a) Total
RNA in solution is passed
through the column in a
high-salt buffer, and the
oligo(dT) tracts bind the
poly(A) tails of the mRNA.
(b) Residual RNA is washed
away with a high-salt buffer,
and (c) the mRNA is eluted
by washing with a low salt
buffer. (d) The mRNA is
then precipitated under
ethanol and collected by
centrifugation.
 HANDLING AND QUANTIFICATION OF
NUCLEIC ACIDS
 Nucleic acid are measured typically in microlitre, nanolitre, or
picograms during a cloning experiment.

 The concentration of a solution of nucleic acid can be determined

by measuring the absorbance at 260 nm, using a
spectrophotometer.

 An A260 of 1.0 is equivalent to a concentration of 50ug ml −1 for

double-stranded DNA, or 40ug ml−1 for single stranded DNA or
RNA. If the A280 is also determined, the A260/A280 ratio
indicates if there are contaminants present, such as residual phenol
or protein. The A260/A280 ratio should be around 1.8 for pure
DNA and 2.0 for pure RNA preparations
 LABELLING OF NUCLEIC ACID
 A major problem encountered in many cloning procedures is
that of keeping track of the small amounts of nucleic acid
involved.

 One way of tracing the material is to label the nucleic acid with
a marker of some sort, so that the material can be identified at
each stage of the procedure. So what can be used as the label?

 Radioactive isotopes are often used to label nucleic acids,

although they are more hazardous than non-radioactive
labelling methods. The most common isotopes used are tritium,
carbon 14, sulphur 35, and phosphorus 32.
 SOME COMMON METHODS OF LABELLING
NUCLEIC ACID MOLECULES
 END LABELLING
 In the end labelling technique, the enzyme polynucleotide kinase is
used to transfer the terminal phosphate group of ATP onto 5’-hydroxyl
termini of nucleic acid molecules. If the ATP donor is radioactively
labelled, this produces a labelled nucleic acid of relatively low specific
activity, as only the termini of each molecule become radioactive

Figure 19: End labelling DNA using polynucleotide kinase (PNK).

 SOME COMMON METHODS OF LABELLING
NUCLEIC ACID MOLECULES CONTD.
 NICK TRANSLATION
 Nick translation relies on the ability of the enzyme DNA polymerase I to translate
(move along the DNA) a nick created in the phosphodiester backbone of the DNA
double helix. Nicks may occur naturally or may be caused by a low concentration of
the nuclease DNase I in the reaction mixture. DNA polymerase I catalyses a strand-
replacement reaction that incorporates new dNTPs into the DNA chain. If one of the
dNTPs supplied is radioactive, the result is a highly labelled DNA molecule.

Figure 20: Labelling DNA by nick translation.

 SOME COMMON METHODS OF LABELLING
NUCLEIC ACID MOLECULES CONTD.
 LABELLING BY PRIMER EXTENSION
 Labelling by primer extension refers to a technique that uses random oligonucleotides
(usually hexadeoxyribonucleotide molecules - sequences of six deoxynucleotides) to
prime synthesis of a DNA strand y DNA polymerase. The DNA to be labelled is
denatured by heating, and the oligonucleotide primers annealed to the single-stranded
DNAs. The Klenow fragment of DNA polymerase can then synthesise a copy of the
template, primed from the 3’-hydroxyl group of the oligonucleotide. If a labelled dNTP
is incorporated, DNA of very high specific activity is produced.

Figure 21: Labelling DNA by primer extension

(oligolabelling).
 SOME COMMON METHODS OF LABELLING
NUCLEIC ACID MOLECULES CONTD.
 NUCLEIC ACID HYBRIDISATION
 The simple base-pairing relationship between complementary
sequences has very far-reaching consequences – both for the cell
and its functioning and for the scientist who wishes to exploit
this feature.

 In addition to providing information about sequence complexity,

nucleic acid hybridization can be used as an extremely sensitive
detection method, capable of picking out specific DNA
sequences from complex mixtures. Usually a single pure
sequence is labelled with Phosphorus 32 and used as a probe.
The probe is denatured before use so that the strands are free to
base-pair with their complements.
 GEL ELECTROPHORESIS
 Separation of biomolecules by gel electrophoresis is one of the most
powerful techniques in molecular biology.

 The method relies on the fact that nucleic acids are polyanionic at neutral
pH; that is, they carry multiple negative charges because of the phosphate
groups on the phosphodiester backbone of the nucleic acid strands. This
means that the molecules will migrate towards the positive electrode when
placed in an electric field.

 As the negative charges are distributed evenly along the DNA molecule, the
charge/mass ratio is constant; thus, mobility depends on fragment length.

 The technique is carried out using a gel matrix, which separates the nucleic
acid molecules according to size.
GEL ELECTROPHORESIS CONTD.

Figure 22: A typical system used for agarose gel electrophoresis. The gel is just
covered with buffer; therefore, the technique is sometimes called submerged
agarose gel electrophoresis (SAGE). Nucleic acid samples placed in the gel will
migrate towards the positive electrode as indicated by the horizontal arrow.
 GEL ELECTROPHORESIS CONTD.
 The type of matrix used for electrophoresis has important
consequences for the degree of separation achieved, which is
dependent on the porosity of the matrix. Two gel types are
commonly used: agarose and polyacrylamide. Agarose is extracted
from seaweed and can be purchased as a dry powder that is melted
in buffer at an appropriate concentration, normally in the range
0.3-2.0% (w/v). On cooling, the agarose sets to form the gel
Table 4: Separation characteristics for agarose and polyacrylamide
gels
GEL ELECTROPHORESIS CONTD.

Figure 23: Black-and-white photograph of an agarose gel,

stained with ethidium bromide, under uv irradiation.
 DNA SEQUENCING
 The ability to determine the sequence of bases in DNA is a
central part of modern molecular biology and provides what
might be considered the ultimate structural information. Rapid
methods for sequence analysis were developed in the late
1970s.

 Two rapid sequencing techniques were developed in the 1970s:

the chemical method and the enzymatic method. Modern DNA-
sequencing technologies are based on the enzymatic method

 DNA sequencing is the process of determining the precise

order of nucleotides within a DNA molecule.
 PRINCIPLES OF DNA SEQUENCING
 The determination of a DNA sequence requires that the
bases are identified in a sequential technique that enables
the processive identification of each base in turn. There
are three main requirements for this to be achieved:

 DNA fragments need to be prepared in a form suitable for

sequencing.
 The technique used must achieve the aim of presenting each
base in turn in a form suitable for identification.
 The detection method must permit rapid and accurate
identification of the bases.
 PREPARATION OF DNA FRAGMENTS
 The strategy employed to tackle large-scale sequencing projects
depends on a number of factors, but essentially comes down to a
choice between the ‘ordered’ and ‘shotgun’ approaches.

Figure 24: Two strategies for sequencing large stretches of DNA. (a) An ordered
approach is shown, (b) The shotgun approach is shown.
 Maxam–Gilbert (chemical) sequencing
 A defined fragment of DNA is required as the starting material. This need not be
cloned in a plasmid vector, so the technique is applicable to any DNA fragment.
The DNA is radiolabelled with Phosphorus 32 at the 5’ ends of each strand, and
the strands are denatured, separated, and purified to give a population of labelled
strands for the sequencing reactions.

 The next step is a chemical modification of the bases in the DNA strand. This is
done in a series of four or five reactions with different specificities, and the
reaction conditions are chosen so that, on average, only one modification will be
introduced into each copy of the DNA molecule.

 The modified bases are then removed from their sugar groups and the strands
cleaved at these positions using the chemical piperidine.

 The theory is that, given the large number of molecules and the different
reactions, this process will produce a set of nested fragments.
Maxam–Gilbert (chemical) sequencing
Contd.

Figure 25: DNA sequencing

using the chemical (Maxam–
Gilbert) method. (a)
Radiolabelled single-
stranded DNA is produced.
(b) The bases in the DNA are
chemically modified and
removed, with, on average,
one base being affected per
molecule. (c) The
phosphodiester backbone is
then cleaved using
piperidine. (d) The process
produces a set of fragments
differing in length by one
nucleotide, labelled at their
5’ termini.
 Sanger–Coulson (dideoxy or enzymatic)
sequencing
 Although the end result is similar to that attained by the chemical method, the
Sanger-Coulson procedure is totally different from that of Maxam and Gilbert. In
this case a copy of the DNA to be sequenced is made by the Klenow fragment
of DNA polymerase.

 The template for this reaction is single-stranded DNA, and a primer must be used
to provide the 3’ terminus for DNA polymerase to begin synthesizing the copy.

 The production of nested fragments is achieved by the incorporation of a

modified dNTP in each reaction. These dNTPs lack a hydroxyl group at the 3’
position of deoxyribose, which is necessary for chain elongation to proceed.

 Such modified dNTPs are known as dideoxynucleoside triphosphates (ddNTPs).

four ddNTPs (A, G, T, and C forms) are included in a series of four reactions,
each of which contains the four normal dNTPs.
Sanger–Coulson (dideoxy or enzymatic)
sequencing contd.

Figure 21: DNA sequencing using the dideoxy chain termination (Sanger–Coulson)
method.
ELECTROPHORESIS AND READING OF
SEQUENCES

Fgure 26: Reading a DNA sequence.

(a) An autoradiograph of part of a
sequencing gel, and (b) a tracing of
the autoradiograph.

Separation of the DNA fragments

created in sequencing reactions is
achieved by PAGE.