Chapter Eight

Molecular Techniques
Outline
 Isolation & extraction of DNA & RNA
 Manipulation of purified DNA
 Recombinant DNA technology/genetic
engineering
 Gene cloning & cloning vectors
 DNA library
 PCR (invitro amplification)
 cont’d
Electrophoresis
Hybridization (insitu hybridization)
RFLP analysis
Blotting
DNA sequencing
Application of molecular biology
 Introduction
 Since the late 1950s & early 1960s, molecular biologists
have tried to characterize, isolate, & manipulate the
molecular components of cells (DNA, RNA & Protein)

1. Isolations & Extraction of nucleic acids: DNA

 The genetic engineer will need to prepare at least three
distinct kinds of DNA:
 Total cell DNA (Genome)
 Plasmid DNA
 Phage DNA
Isolation & Extraction of Total cell DNA
1. Growth & harvesting
 A culture of bacteria is grown & harvested in a
liquid (broth) medium

2. Preparation of cell extract

 since DNA is inside the cell, cells envelop must be
broken to release their contents
 Cells envelop: cell wall (rigid) & cell membrane
 2.1. Cell lysis
Cell wall:
 Lysozyme : breaks cell wall of gram negative bacteria
 EDTA : removes Mg 2+ by cheletion, because Mg2+ is a
cofactor for most endonuclease & is an important
component of cell envelop.
 NaCl: keeps cells happy & prevent unwanted aggregation
Cell membrane
 Disrupt cell membrane so that the DNA is released in to the
extraction buffer.
 SDS (sodium dodecyl sulphate) lyses cell membrane by
removing lipid molecules from cell membrane.
 CTAB ( cetyletrimethyl ammonium bromide) remove
carbohydrates
2.2. Purification of cell extract

 Removal of contaminants: Protein & RNA

a. Centrifugation: removal of cell debris
b. Ribonuclease treatment: RNA removal
c. Deproteinization: Protein removal
 Protein can be removed by phenol, or, 1:1

phenol: chloroform, or phenol: chloroform:

isoamyl alcohol (24:23:1)
2.3 Precipitation of DNA
A common procedure is to use 0.3 M sodium acetate
followed by ethanol.
Addition of cold alcohol makes the DNA clump together
& precipitates out of solution.

2. 4 DNA quantification & purity

Quantification:
DNA & RNA can be quantified by measuring the
absorbance at 260 & 280 nm
A value of 1.0 in a 1 cm path length corresponds to a
concentration of 40 μg/mL for RNA & 50 μg/mL for DNA
Manipulation of purified DNA
 Cutting & joining are the two examples of DNA
manipulation to lengthen or shorten DNA molecule
 Manipulation provide foundation for gene cloning,
basic studies in biochemistry & control of gene
expression
 1. Manipulative enzymes
DNA ligase
Join nucleic acid molecules by making phosphodiester
bonds

DNA polymerase I:
is an enzyme that has a dual activity; polymerization and
degradation
These activities are controlled by different parts of the
enzyme molecule contained in the first 323 amino acids of
the polypeptide.
Klenow fragments
o If you remove these 323 amino acids, then there will
be only polymerase activity & is unable to degrade
DNA
o This modified enzyme is called Klenow fragments.

Reverse transcriptase
o It is the enzyme that involved in the replication of
several kinds of viruses
o It uses RNA as a template not DNA
o It is used for cDNA cloning.
 cont’d
DNA modifying enzymes
 Are enzymes that modify molecules by addition or
removal of specific chemical groups
Alkaline phosphatase (from E. coli)
 Remove phosphate group from 5’ termini of DNA

Polyucleotide kinase (from E. coli)

 Have a reverse effect to alkaline phosphatase,

Terminal deoxynucleotide transferase

 obtained from calf thymus
 Add one or moe deoxynucleotide on to the 3’-
terminus of DNA molecule
 Nucleases
 Nucleases are the enzymes that break the
phosphodiester bonds (that hold nucleotides
together) of DNA.
 Two types: Endonuclease and Exonuclease

A. Endonucleases: it breaks internal phosphodiester bonds

within a DNA molecule
 They are called restriction endonucleases because their
function in the bacteria restrict incoming foreign DNA
 They recognize different sequences of bases on a DNA
molecule called palindromes & cuts at a specific sites
called restriction sites producing sticky or blunt ends.
B. Exonucleases: are enzymes which removes nucleotide
one at a time from the end of a DNA molecule
Restriction Endonuclease/Restriction Enzyme
They are isolated from bacteria and received their name
because they restrict or prevent viral infection by
degrading the invading viral DNA
are the cornerstone of recombinant DNA technology
They recognize a specific nucleotide sequence and cut
both strands of the DNA within that sequence.
There are two types of RE

1.Type I restriction enzymes: cut the DNA a thousand or

more base pairs away from the recognition site
2.Type II restriction enzymes: cut the DNA in the middle
of the recognition site. E.g., Sau3A, EcoRI, etc.
Some of Restriction enzymes
Restriction enzyme (RE) digestion
 Restriction enzyme recognition sites are usually four,
six or eight bases long and the sequence forms an
inverted repeat.
 RE type-II can cut DNA in two different ways.
A. Many make a simple double-stranded cut, giving a
sequence called blunt or flush ends.
B. Others cut the two strands at d/t positions, usually
just a few nucleotides apart, resulting short single-
stranded overhangs, called sticky or cohesive ends
 DNA fragments with sticky ends are particularly useful
for recombinant DNA technology. This is because the
single-stranded sticky DNA ends can easily pair with
any other DNA fragment having complementary sticky
ends.
Characteristics of restriction enzymes:
1. Cut DNA sequence-specifically
2. They are bacterial enzymes
3. Named form bacterial genus, species, strain, and type
(e.g., EcoRI)
4. Recognize specific 4-8 bp sequences
 sequences have symmetry (they are palindromes)
 after cutting the DNA, the cut ends are either blunt
or sticky ends
5. Frequency of cutting
 4-base cutter 44 = 256 bp
 6-base cutter 46 = 4,096 bp
 Recombinant DNA technology/genetic
Engineering & gene cloning
 Cloning is making of identical copies. DNA cloning is
process of making several identical copy of a gene or
gene fragment.
 DNA fragment from an organism is cleaved or
amplified and inserted in a DNA carrier called vector.
 Vectors are generally double stranded circular DNA
which has origin of replication through which they can
replicate in the host system.
 Vectors also have a selectable marker (generally
antibiotics resistance gene) for screening of recombinant
colonies.
 Cont’d
Vector with desired DNA insert is called recombinant
DNA.
This can be transferred to suitable host system (generally
E.Coli) where it finds machinery for replication and makes
several copies of it (may also express protein).
The process is also called recombinant DNA technology
or genetics engineering. .
Recombinant DNA technology is the use of in vitro
molecular techniques to isolate and manipulate fragments
of DNA
Recombinant DNA technology and gene cloning have
been fundamental to our understanding of gene structure
and function
 Recombinant DNA refers to the creation of new
combinations of DNA segments that are not found
together in nature. The isolation and manipulation of
genes allows for more precise genetic analysis as well as
practical applications in medicine, agriculture, and
industry.
 Gene cloning is the incorporation of a foreign gene into
a vector to produce a recombinant DNA molecule that
replicates & expresses the foreign gene in a recipient
cell.
 They have many medical applications, including
development of new vaccines, biologics, diagnostic
tests, & therapeutic methods
Gene cloning: in-vivo amplification of DNA
 Genes from other species, even from eukaryotic cells
can be introduced into bacterial cells by genetic
manipulation & subsequently cultured forming a clone
of genetically identical cells.
 Each cells contains the foreign gene that has been
duplicated with the normal bacterial genes, such
techniques is called gene cloning.
 The two molecules that are required for cloning are the
DNA to be cloned & a cloning vector.
Cloning vector
is a DNA molecule that carries foreign DNA into a host
cell, replicates inside a bacterial (or yeast) cell &
produces many copies of itself & the foreign DNA
Requirements for a cloning vector

¨ Should be capable of replicating in host cell

¨ Should have convenient RE sites for inserting DNA
of interest
¨ Should have a selectable marker to indicate which
host cells received recombinant DNA molecule
¨ Should be small and easy to isolate
a number of vectors currently in use, which permits the
cloning of DNA fragments over a wide size range.
Some of Cloning Vectors
Plasmids: extra chromosomal circular double stranded
DNA replicating elements present in bacterial cells.
 Cloning limit: 100 to 10,000 bps

Phage: These are intracellular obligate parasites that

multiply inside bacterial cell and have a very high
significant mechanism for delivering its genome into
bacterial cell
 Cloning limit: 8-20 kb.

Cosmids
 an extra-chromosomal circular DNA molecule that
combines features of plasmids & phage
 cloning limit: 35-50 kb
Bacterial Artificial Chromosomes (BAC)
 cloning limit: 75-300 kb
 very cost effective
Yeast Artificial Chromosomes (YAC)
 an artificial chromosome that contains telomeres,
origin of replication, a yeast centromere, & a
selectable marker for identification in yeast cells;
 cloning limit: 100-1000 kb
Gene cloning Steps
 prepare the vector & DNA to be cloned by
digestion with RE
 ligate the foreign DNA into the vector with DNA
ligase
 Introduce the DNA into bacterial cells (or yeast
cells for YACs) by transformation
 select cells containing foreign DNA by
screening for selectable markers
 May 1, 2025
32
Clone Identification

• Probing for correct DNA sequence

• Probing for correct protein product

• Antibiotic resistance
Selection: Nucleic Acid Hybridization
 Among the uses of cloned DNA are:
 DNA sequencing. All current methods of DNA
sequencing require cloned DNA fragments.
 Nucleic acid hybridization. an important
application of cloned DNA fragments entails
incorporating a radioactive or light-emitting “label”
into them, after which the labeled material is used
to “tag” DNA fragments containing similar
sequences.
 Storage and distribution. Cloned DNA can be
stored for long periods without risk of change and
can easily be distributed to other researchers.
DNA library

 It is a collection of cloned DNA fragments

 Two types:

1. Genomic &

2. cDNA libraries
Genomic DNA library
 Genome is a collection of clones of all the
DNA sequences of a given species

 It can be prepared into two different ways

using two different vectors, plasmid or
lambda phage.
Genomic Library
Genomic library using plasmid vector

 Plasmids have 3 kinds of regions

 insertion region

 origin of replication

 R genes – antibiotic, radiation, &

heavy metals
Steps of genomic library:
 preparation of plasmid vector
 extraction of DNA of interest
 insertion of a DNA fragment to a plasmid
vector
 Cloning in a bacterial cells
Genomic library using lambda phage
 preparation of lambda phage
 preparation of genomic DNA fragment
 hybridization of genomic DNA fragment with lambda
arms
 preparation of infectious lambda virion carrying
recombinant lambda phage
 Finally, the recombinant lambda virion are plated on a
lawn of bacteria, E. coli
 Since each plaque arises from a single recombinant virion,
all the progeny λ phages that develop are genetically
identical & constitute a clone carrying a particular
genomic DNA of interest
May 1, 2025
cDNA library
 Large collection of cDNA copies of all the
mRNA in a cell type
 Represent sequences expressed as mRNA in a
particular cell type
 Lack non-coding introns
 DNA that is made from RNA is called
complementaryDNA (cDNA)
 It could be single- or double-stranded
Steps of cDNA Preparation
 Isolation of mRNA from the cell
 synthesis of cDNA from each mRNA
 conversion of single mRNA to double stranded cDNA
 incorporation of cDNA into a vector (plasmid or
phage)
 ligation of the restriction cleaved ds cDNA to plasmid
or lambda phage
 plating recombinant vector on lawn of E. coli to
produce a library
Isolating RNA
cDNA library construction, plasmid
cDNA library construction, phage
Polymerase Chain Reaction (PCR):
an invitro amplification
 PCR is a laboratory (in vitro) technique for generating
large quantities of DNA
 PCR is a cell-free amplification technique for
synthesizing multiple identical copies (billions) of any
DNA of interest
 The PCR was developed in 1986 by Kary Mullis, who
won noble price in chemistry in 1993 for developing
the PCR technique
Applications of PCR:
1. Cloning genes encoding a known protein
2. Diagnostic (detection of bacterial & viral infection)
3. DNA finger printing & forensics
4. Making probes: Amplified product can be used as a
probe to pull out the full length gene from a cDNA or
genomic library
5. Amplifying 'old DNA‘
6. Amplifying cloned DNA from vectors
7. Genetic diagnosis: diagnosing inherited disorders and
cancer
8. Blood group typing
9. Prenatal diagnosis - such as determining the sex of
fetuses for those at risk of X-linked disorders
The components involved in the polymerase chain
reaction are as follows:
1. Template DNA, that is to be copied = 0.2 -1 μg
2. Two PCR primers to initiate DNA synthesis.
3. DNA polymerase:
 Heat resistant DNA polymerase is required.
 Taq polymerase from Thermus aquaticus is most
widely used.
 Isolated from heat resistant bacteria living in hot
springs at T0 up to 90°C
4.Nucleoside triphosphates (NTPs)- to supply
nucleotides.
5. PCR machines (sometimes called thermocyclers).
 Steps of PCR
 PCR amplification of DNA occurs by repeated cycles
(25-30) of three temperature dependent steps
1. Denaturation
 The dsDNA template is denatured by briefly heating
the sample to 92-940C ( for 5 min).
2. Annealing:
 When the temperature is lowered to 45–55°C (30 sec),
the primers can anneal to the ends of the target sequence
 To ensure adequate specificity, the primers must be 20-
30 nucleotides long.
 Since the primer is present in large excess over the
template DNA, essentially all template strands will bind
to primers rather than reannealing to each other.
3. Primer extension
(Polymerization):
 Once the primers
have annealed to
the template, the
T0 is increased to
60-75°C.
 the thermostable
Taq polymerase
extends each primer in
the 5' to 3' direction
 The rate of extension is
about 50-100 nts/sec.
Thus, the time required
for primer extension
depends on the length of
the sequence to be
amplified.
PCR
 DNA between the primers doubles with each thermal
cycle

Number
1 2 4 8 16 32 64

0 1 2 3 4 5 6
Cycles
Theoretical yield
 It is 2n x Y
 Where:
Y = the starting number of copies &
n = the number of thermal cycles
E.g. If you start with 100 copies, how many
copies are made in 30 cycles?
o 2n x Y = 230 x 100 = 107,374,182,400
Advantage of PCR
DNA from one cell equivalent can be
amplified
PCR products can be directly sequenced
PCR products can be directly cloned
DNA sequences up to 30 kb can be
amplified
62 May 1, 2025
 Controls for PCR
 Blank reaction
 Controls for contamination
 Contains all reagents except DNA template
 Negative control reaction
• Controls for specificity of the amplification reaction
• Contains all reagents and a DNA template lacking
the target sequence
 Positive control reaction
 Controls for sensitivity
 Contains all reagents & a known target containing
DNA template
 Gel Electrophoresis
 The DNA fragments produced by a restriction
enzyme can be separated by size using the fact
that DNA is negatively charged and moves in
response to an electric field.
 The movement of charged molecules in an electric field
is called electrophoresis.
 The type of electrophoresis most commonly used in
Molecular Biology is gel electrophoresis.
Application of electrophoresis
 To determine the presence or absence of PCR products
 To quantify the size of the product
 Separating DNA of different Lengths
 Instrumentation & reagents of
electrophoresis
1. Gel casting apparatus
a. Casting tray
 It serves as the mold that will provide the shape
& size for the gel
b. Comb
 Sample combs, around which molten agarose is
poured to form sample wells in the gel.
c. Support
 The support prevents the liquid agarose from
leaking out of the mold during the solidification
process.
2. Support medium
 It is a medium on which separation takes place
 It may be paper or gel made up of agarose, cellulose
acetate, starch, or polyacelamide

Agarose gels
 consisting of two galactose-based polymers, agarose
& agaropectin.
 Purified agarose is generally purchased in a
powdered form & made by dissolving the dry
polymer in boiling buffer.
3. Buffers
 Buffer serve as: carries the applied current, maintain
constant PH and determine electrical charge on the
solute.
 Types of buffers: Usually Tris-acetate-EDTA (TAE)
or Tris-borate-EDTA (TBE).
4. Stains
 Ethidium bromide : a fluorescent dye used for
staining nucleic acids.
5. Transilluminator (Uv-light box)
 Used to visualize EtBr-stained DNA in gels.

6. Power supplies: It is a deriving force

Electrophoresis procedure
A. gel preparation
 Mini-gel - 40 ML and Medium-gel - 100 ML.

B. Casting agarose gel

 Set gel-casting tray into the tray apparatus
(Perspex plate), screw tight, & insert well-forming
comb.
 Place tray FLAT where agarose can be poured &
allowed to set UNDISTURBED.
 Pour 40ml of agarose solution (liquefied in 60 0C)
into casting tray.
 Cont’d
Gel will become cloudy as it solidifies (20 minutes).
Do NOT move tray while agarose is solidifying.
Gently remove comb, pulling it straight up & taking
care not to rip wells.
When solidified, remove the gel tray from the gel-
casting tray & place on platform electrophoresis box,
the comb at negative (BLACK) cathode end..
Fill box with TBE buffer to a level that just covers
entire surface of gel by about 2mm.
Make certain that sample wells left by comb are
completely submerged by buffer.
The gel is now ready to load with DNA.
C. Loading gel with DNA
 Remove the tubes from the fridge & tap the tubes
firmly down on the table top
 Add 2 μl loading dye to each reaction tube & tap
contents of tube on table top.
 Use pipette to load contents of each reaction tube into
a separate well in gel
 Set your micropipetter on 12 μl (total contents in the
tube).
Fig. loaded gel electrophoresis
D. Run electrophoresis
 Place the chamber top on the chamber, connect
electrodes to power supply.
 Power supply is turned on & voltage set at
120V.
 In a few minutes, you should begin to notice
the loading dye moving through the gel toward
the + pole (anode).
 Let it run for about 1 hour.
 Cont’d
The loading dye will resolve into 2 bands of color. The
faster-moving, purplish band is the dye bromophenol
blue, the slower-moving, aqua band is xylene cyanol
After 1 hour, the bromophenol band should be nearing
the end of the gel.
Turn off the power supply, disconnect the leads, &
remove the top of chamber.
Carefully remove the casting tray & keep it horizontal
until you are ready to put into the plastic container.
Add enough methylene blue DNA stain to cover the gel
& place cover on it.
Gel Electrophoresis
Run Gel Uv light box
F. INTERPRETATION:
 The sizes of the various fragments can be
identified by including a “ladder” in the gel
 A ladder is a mixture of DNA fragments of
known size
 It is usually run beside the unknown sample
so that the sizes of various DNA fragments in
the sample can be identified
Sizing a Gel Product

Base
Pairs
(bp)
4000
3000
2000 bp
2000
1600 1000 bp
1000

500

1Kbp Sample
ladder
Factors affecting electrophoresis
 The electric field: an increase in voltage will
therefore increase the rate of migration
proportionally
 Size of DNA: the rate of migration decreases
for larger molecules, due to the increased
frictional & electrostatic forces exerted by the
surrounding medium.
Concentration of the gel
•By increasing the agarose concentration the
smaller DNA fragments will give a clearer
separation
•By lowering the agarose concentration the
larger fragments of DNA will give a clearer
separation
•By optimizing the % agarose one can clearly
separate a mixture of similar DNA fragments
 Nucleic Acid Hybridization
 Most genomes are sufficiently large and complex that
digestion with a restriction enzyme produces many
bands that are the same or similar in size.
 The DNA fragments in a gel are usually immobilized by
transferring them onto a sheet of special filter paper
consisting of nitrocellulose, to which DNA can be
permanently bound.
 a double helix can be “unzipped” to form single strands
and two single strands that are complementary or nearly
complementary in sequence can be “zipped” together to
form a different double helix.
 The “unzipping” is called denaturation, the “zipping”
renaturation.
 Cont’d
Denatured DNA strands can form double-stranded DNA
with other strands, provided that the strands are sufficiently
complementary in sequence.
This process of re-naturation is called nucleic acid
hybridization because the double-stranded molecules are
“hybrid” in that each strand comes from a different source.
For DNA strands to hybridize, two requirements must be
met:
 The salt concentration must be high to neutralize the
negative charges of the phosphate groups
 The temperature must be high enough to disrupt
hydrogen bonds
 Blotting
 The porous & thin nature of a gel is ideal for separating
DNA fragments using electrophoresis, but these gels are
delicate & rarely usable for other techniques.
 For this reason, DNA that has been separated is
transferred from a gel to an easy-to-handle inert
membrane by a process called blotting.
 The term "blotting" describes the overlaying of the
membrane on the gel & the application of a pad to ensure
even contact, without disturbing the positions of the
DNA fragments
 Types: southern, northern & western
Southern blotting= DNA:DNA probe
hybridization
The methods of DNA cleavage,
electrophoresis, transfer to nitrocellulose, and
hybridization with a probe are all combined in
the Southern blot
The technique was developed by Edward
Southern in 1975.
It is used to detect the presence of a
particular piece of DNA in a sample.
Southern blot: Procedure
 In this procedure, a gel in which DNA molecules have
been separated by electrophoresis is treated with alkali
to denature the DNA and render it single-stranded.
 Then the DNA is transferred to a sheet of
nitrocellulose in such a way that the relative positions
of the DNA bands are maintained.
 The nitrocellulose, to which the single-stranded DNA
binds tightly, is then exposed to denatured radioactive
complementary DNA (the probe) in a way that
leads the complementary strands to anneal to
form duplex molecules.
 Radioactivity becomes stably bound (resistant to removal
by washing) to the DNA only at positions at which base
sequences complementary to the radioactive molecules
are present, so that duplex molecules can form.
 The radioactivity is located by placing the paper in
contact with x-ray film; after development of the film,
blackened regions indicate positions of radioactivity.
 For example, a cloned DNA fragment from one species
may be used as probe DNA in a Southern blot with DNA
from another species; the probe will hybridize only with
restriction fragments containing DNA sequences that are
complementary enough to allow stable duplexes to form.
Southern Blotting
2. Northern blotting= RNA:DNA
hybridization
 Northern blotting is a simple extension of Southern
blotting & named because it is patterned after earlier
technique.
 It is used to detect cellular RNA rather than DNA.
 Initially, it was thought that RNA would not bind
efficiently to filter however, it was then shown that
when RNA was denatured, that it would also bind
efficiently to nitrocellulose.
Purpose of Northern blotting
 The quantity of the mRNA present.
 Whether a genomic DNA probe has regions that are
transcribed
Western blotting = Ag : Ab hybridization
It is also called immunoblot, is similar to
southern & northern blot
It is used to detect Abs to specific of
elctrophoretically separated subspecies of Ags
(protiens)
 Types of DNA Markers Present in
Genomic DNA
 Genetic variation, in the form of multiple alleles of
many genes, exists in most natural populations of
organisms.
 such genetic differences between individuals are
called DNA markers; they are also called DNA
polymorphisms. (The term polymorphism literally
means “multiple forms.”)
 There are some of the principal methods for
detecting DNA polymorphisms among individuals.
Single-Nucleotide Polymorphisms (SNPs)
SNP is present at a particular nucleotide site if the DNA
molecules in the population frequently differ in the identity of the
nucleotide pair that occupies the site.
For example, some DNA molecules may have a T-A bp at a
particular nucleotide site, whereas other DNA molecules may have
a CG base pair at the same site. This difference constitutes a SNP.
The SNP defines two “alleles” for which there could be three
genotypes among individuals in the population:
homozygous with T-A in both homologous chromosomes,
homozygous with CG in both homologous chromosomes,
or heterozygous with TA in one chromosome and CG in the
homologous chromosome.
 RFLP (Random fragment length polymorphism)
 A SNP that eliminates a restriction site is known as a
restriction fragment length polymorphism.
 Because RFLPs change the number and size of DNA
fragments produced by digestion with a RE, they can be
detected by the Southern blotting.
 E.g. SNP consists of a T-A bp in some molecules and a C-
G bp in others. In this e.g., the polymorphic nucleotide
site is included in a cleavage site for the RE EcoRI (5'-
GAATTC-3').So, DNA molecules with T-A at the SNP
will be cleaved at the middle site, yielding two EcoRI
restriction fragments. Alternatively, DNA molecules with
C-G at the SNP will not be cleaved at the middle site (b/c
the presence of C-G destroys the EcoRI restriction site)
and so will yield only one larger restriction fragment.
Restriction Fragment Analysis
 DNA sequencing
 The process of determining the order of the nucleotide
bases along a DNA strand is called sequencing.
 During the 1970s two DNA sequencing methods were
devised
 One method, developed by Allan Maxam and Walter
Gilbert, involves the base-specific cleavage of DNA
 The other method, developed by Frederick Sanger, is
known as dideoxy sequencing
 The dideoxy method has become the more popular and
will therefore be discussed here
Sanger (dideoxy) method
 The most popular method for doing sequencing is
called the dideoxy method or Sanger method.
 named after its inventor, Frederick Sanger, who was
awarded the 1980 Nobel prize in chemistry [his second]
for this achievement.
 DNA is synthesized from four deoxynucleotide
triphosphates. Each new nucleotide is added to the 3′ -
OH group of the last nucleotide added.
 The dideoxy sequencing method employs DNA
synthesis in the presence of small amounts of
nucleotides that contain the sugar dideoxyribose
instead of deoxyribose (Fig below).
 Dideoxyribose lacks the 3'-OH group, which is
essential for attachment of the next nucleotide in
a growing DNA strand
 so incorporation of a dideoxynucleotide instead of
a deoxynucleotide immediately terminates further
synthesis of the strand.
 For this reason, the dideoxy method is also called the
chain termination method.
 Procedure
 To sequence a DNA strand, four DNA synthesis
reactions are carried out.
 Each reaction contains:
 the single-stranded DNA template to be sequenced,
 a single oligonucleotide primer complementary to a
stretch of the template strand,
 all four deoxyribonucleoside triphosphates, and
 a small amount of one of the nucleoside triphosphates
in the dideoxy form.
 Each reaction produces a set of fragments that terminates
at the point at which a dideoxynucleotide was randomly
incorporated in place of the normal deoxynucleotide.
 Therefore, in each of the four reactions, the lengths of
the fragments are determined by the positions in the
daughter strand at which the particular
dideoxynucleotide present in that reaction was
incorporated. The sizes of the fragments produced by
chain termination are determined by gel
 Electrophoresis, and the base sequence is then
determined by the following rule:
 If a fragment containing n nucleotides is generated
in the reaction containing a particular
dideoxynucleotide, then position n in the daughter
strand is occupied by the base present in the
dideoxynucleotide. The numbering is from the 5'
nucleotide of the primer.
How to read?
How to interpret ssDNA to be
sequenced
Application of molecular biology

 Help to diagnose genetic diseases

 Provide better & specific diagnosis of microbial
infections
 To determine paternity right decision
 As full proof method to identify people incase of
rapes
 identifying new genes & the protein they encode
 to correct endogenous genetic defects
 to manipulate large quantities of specific gene
products such as hormones & vaccines