Recombinant DNA Technology 22.ppt

Recombinant DNA
Technology
Dr. Dharmveer Sharma
Professor (D)
Department of Biochemistry,
Govt. Medical College, Shivpuri (M.P.)
Email Id:biokem123@gmail.com
Mob. No. 08115005237

 Recombinant DNA technology came into existence
in the 1970s, and has revolutionised biochemistry
 As the name suggests, recombinant DNA is
formed by joining two (or more) different DNA
molecules or fragments of DNA molecules
 Such DNA, having unrelated genes, is known as
chimeric DNA, named after a mythological
creature, chimera having the head of a lion, the
trunk of a goat and the tail of a snake

 Recombinant DNA technology has proved to be
of immense value in :
• Medicine
• Agriculture
• Animal husbandry
• Industry

 
Reverse
transcriptase
mRNA-cDNA hybrid

Hydrolysis
of mRNA
cDNA

Insulin gene (exons)
DNA ligase

Nicked
plasmid
Plasmid with
insulin gene
Introduction
into E. coli

Pancreas
E.coli with insulin gene
mRNA for
insulin
DNA
polymerase
Synthesis of recombinant human insulin

Most common enzymes use in
RDT

Recombinant DNA Technology 22.ppt

RESTRICTION ENDONUCLEASES
 Restriction endonucleases (or restriction
enzymes) are among the most important
tools of recombinant DNA technology
 In fact, it is was the discovery of restriction
enzymes by Arber, Smith and Nathans in the
early 1970s that opened the doors of
recombinant DNA research
Arber Smith Nathans

 Restriction enzymes are found in bacteria, and
protect the bacterial cells against viral infections
 When a virus infects a bacterial cell, viral DNA is
split by bacterial restriction enzymes and, thus,
the virus is destroyed

 Hundreds of restriction enzymes have been
discovered so far
 These are named after the bacterium in which
they are found e.g. Hin I, Hae III, Eco RI etc
 The three-letter abbreviation stands for the
bacterium e.g. Hin for Haemophilus influenzae,
Hae for Haemophilus aegyptius, Eco for
Escherichia coli

 Sometimes, a strain designation is also included
in the name e.g. R after Eco means strain R of
E.coli
 The numerals I, II, III etc indicate the serial
numbers of the enzymes from the same
bacterium in the order of discovery

 Each restriction enzyme recognises and splits a
specific base sequence in double-stranded DNA
 The base sequence recognised by a restriction
enzyme extends over 4-8 base pairs, and is
palindromic (a palindrome is a word or a
sentence which reads the same from left to
right and from right to left e.g. DAD, ABBA,
MADAM, RADAR etc)

 In DNA, the base sequences are read from 5’ to
3’ direction
 If a sequence reads the same on both the
strands from 5’ to 3’ direction, it is known as a
palindromic sequence

 Restriction enzymes split both the strands
producing either blunt (even or non-
overlapping) ends or sticky (overlapping or
cohesive) ends
 Sticky ends are more useful in recombinant DNA
technology as these can be easily ligated to the
complementary sticky ends of another fragment
of DNA produced by the same restriction enzyme

5’ GG CGCC 3’
5’ GG CGCC 3’
3’ CC GCGG 5’
3’ CCGC GG 5’
5’ GG
3’ CC
CGCC 3’
GCGG 5’
+
5’ GG
3’ CCGC
CGCC 3’
GG 5’
+
RE
Blunt ends produced by a restriction enzyme (RE)
Sticky ends produced by another RE


RE

 The site cleaved by a particular restriction
enzyme is known as its restriction site
 The DNA fragments produced by restriction
enzymes are known as restriction fragments
 A DNA, generally, has several restriction sites for
a number of restriction enzymes

5’ – – G AATTC – – 3’
3’ – – CTTAA G – – 5’
5’ – – G GATCC – – 3’
3’ – – CCTAG G – – 5’
5’ – – GG CC – – 3’
3’ – – CC GG – – 5’
5’ – – G AATTC – – 3’
3’ – – CTTAA G – – 5’
5’ – – G GATCC – – 3’
3’ – – CCTAG G – – 5’
5’ – – GG CC – – 3’
3’ – – CC GG – – 5’
Eco RI
Bam HI
Hae III


 +
+
+







RECOMBINANT DNA
 Restriction fragments obtained from different
sources can be joined together by DNA ligase
to produce recombinant DNA having a base
sequence different from that of the original DNA
 For example, a foreign gene can be inserted into
DNA by this method

5’ – – GGATCC – – 3’
3’ – – CCTAGG – – 5’
5’ – – GGATCC
3’ – – CCTAGG
GGATCC – – 3’
CCTAGG – – 5’
Foreign gene
DNA having Bam HI
restriction site Foreign gene having Bam HI restriction site on either side

Bam HI

Bam HI
Foreign gene inserted between restriction
fragments by ligating complementary sticky ends
5’ – – GGATCC
3’ – – CCTAGG
GGATCC – – 3’
CCTAGG – – 5’
Foreign gene
– – – – – – – –
– – – – – – – –
5’ – – G GATCC – – 3’
3’ – – CCTAG G – – 5’
5’ – – G GATCC G GATCC – – 3’
3’ – – CCTAG G CCTAG G – – 5’
Foreign gene
– – – – – – – –
– – – – – – – –
DNA ligase
+ + +

– – – – – – – –
– – – – – – – –

 After preparing recombinant DNA, amplification
is often required to prepare multiple copies of
the DNA
 Amplification can be done either by cloning the
DNA in some living cell or by polymerase chain
reaction in a test tube

CLONING OF RECOMBINANT DNA
 Cloning of DNA was pioneered by Herbert Boyer
and Stanley Cohen
Herbert Boyer Stanley Cohen

CLONING OF RECOMBINANT DNA (Contd.)
 A clone is a population of identical organisms or
cells or molecules derived from the same source
 Recombinant DNA can be cloned in living cells
using suitable vectors to transfer the
recombinant DNA into the cells
 Some useful cloning vectors are plasmids,
bacteriophages, cosmids and yeast artificial
chromosomes

Plasmids
 Many prokaryotes possess a small circular
double-stranded DNA in addition to the
chromosomal DNA
 This extra-chromosomal DNA is known as a
plasmid
 Plasmids may replicate independently of the
chromosomes

 One or more antibiotic-resistance genes are
often present in plasmids which provide
antibiotic resistance to the micro-organisms
 Bacterial plasmids can be transferred from one
bacterium to another
 DNA fragments upto 10 kb (kilobases) in size
can be easily inserted into plasmids, and cloned
in bacteria e.g. E.coli

 The plasmid is nicked by a suitable restriction
enzyme to generate sticky ends
 The foreign DNA having complementary sticky
ends is joined to the nicked plasmid by DNA
ligase
 The plasmid is introduced into a bacterial cell
 Multiplication of the plasmid and the bacterial cell
produces multiple copies of the foreign gene


Plasmid with
foreign DNA

Plasmid
 
Restriction site
Restriction
enzyme
DNA ligase
Nicked
plasmid
Foreign
DNA
Introduction
into a
bacterial cell
Cell
division
Plasmid Chromosome
Bacterial cell

 pSC 101 and pSC 102 is a plasmid naturally
present in E. coli
 It was the first vector used for cloning
 It has only one restriction site , only one antibiotic-
resistance gene and replicates poorly
 An ideal vector should replicate rapidly, should
have several restriction sites for different
restriction enzymes and more than one
antibiotic-resistance genes

 Several plasmid vectors having these
characteristics have been constructed in the
laboratory e.g. pBR 322- pBR 325,
pBR328, pBR 329, pUC18 etc (pBR- Bolivar
and Rodriguez)
 Plasmids can be introduced into bacteria by
exposing them to high concentration of
divalent cations which increase the
permeability of bacterial cell membrane so
as to allow plasmids to enter the bacterial cells

 However, plasmids may not enter all the
bacterial cells and some plasmids might not
have taken up the foreign DNA
 To select bacterial cells harbouring
recombinant plasmids, use may be made
of antibiotic-resistance genes

 For example, plasmid pBR322 possesses genes
for ampicillin-resistance as well as
tetracycline-resistance
 Each of these two genes has a number of
restriction sites
 A foreign gene (or a DNA fragment) can be
inserted in the middle of, say, tetracycline-
resistance gene

Genetic map of plasmid cloning vector pBB322

Bacteriophages
 Bacteriophages are viruses that infect bacteria
 These viruses have a DNA genome surrounded
by a protein coat

The virus infects a bacterial cell by injecting its DNA into the
bacterial cell – Phase ƛ and Phase M13

 After entering the bacterial cell, the viral DNA
may enter one of the two alternate pathways:
1. Lysogenic pathway
2. Lytic pathway

Lysogenic pathway
 In this pathway, the viral DNA gets incorporated
into the bacterial genome, and becomes
dormant (provirus)
 The proviral DNA replicates only when the
bacterial cell divides

Lytic pathway
 In the lytic pathway, the viral DNA remains
separate
 It replicates independently

Cosmids
 Cosmids are hybrids of plasmids and lambda
phage
 Lambda phage DNA possesses sticky ends
known as cos sites on either side
 These cos sites are necessary for packaging the
phage DNA into the protein coat

 Cosmids are prepared by inserting the cos sites
of phage DNA in plasmids
 Cosmids can infect E.coli just like plasmids
 DNA fragments up to 45 kb in size can be
inserted in cosmids

Yeast Artificial Chromosomes
 Yeast artificial chromosomes (YACs) are linear
double-stranded DNA molecules having the
genes necessary for replication
 Large pieces of foreign DNA (up to 3,000 kb) can
be inserted in YACs, and cloned in yeast cells

METHODS OF GENE TRANSFER
 Transformation- uptake of plasmid DNA by E. coli is
carried out in ice-cold CaCl2 ( 0-5°C), and a
subsequent heat shock (37-45°C for about 90 sec)
 Conjugation- is a natural microbial recombination
process
 Electroporation- based on the principle that high
voltage electric pulses can induce cell plasma
membranes to fuse
 Liposome-mediated gene transfer- Liposomes are
circular lipid molecules, which have an aqueous
interior that can carry nucleic acids

POLYMERASE CHAIN REACTION
 Polymerase chain reaction (PCR) is a technique
for amplification of DNA devised by Mullis(1984)
Kary Mullis

 This technique is far quicker, easier and
inexpensive than cloning
 The only limitation of PCR is the size of DNA
that can be amplified
 DNA up to 3 kb in length can be amplified by
PCR

 In PCR, the DNA to be amplified is replicated by
DNA polymerase of Thermus aquaticus (Taq), a
bacterium found in hot water springs
 The optimum temperature of Taq polymerase is
72°C, and it is not denatured at temperatures upto
95°C
 This property is important because DNA has to
be heated to 94°-95°C for separation of strands,
and Taq polymerase is not destroyed at this
temperature

 For amplifying a desired sequence in DNA, we
have to know short flanking sequences on
either side of the target sequence so that
complementary primers can be prepared
 The reaction mixture contains the DNA to be
amplified, a large quantity of primers and
deoxyribonucleotides (dATP, dGTP, dCTP and
dTTP), and Taq polymerase

 Amplification occurs in three steps:
• The temperature is raised to 95°C for a short
period (30 sec) so that the two strands of DNA
separate
• The temperature is lowered to 55°C for a short
period (30 sec) so that primers can bind to
complementary sequences on both the strands of
DNA
• The temperature is raised to 75°C for 2 to 5 min so
that Taq polymerase can replicate the two strands

Double-stranded DNA with the target sequence
to be amplified
The two strands separate.
Primers hybridise with template strands.
Taq polymerase replicates both the strands.
Two copies of target DNA are formed.
Second cycle begins. Both the double-stranded
DNA molecules are separated into single strands.
Primers bind to all the four strands.
Taq polymerase replicates all the four strands.
Four copies of target sequence are formed.


Target sequence
5´ 3´
95°C for 30 sec

56°C for 30 sec
72°C for 2 to 5 min
72°C for 2 to 5 min
3´ 5´
5´ 3´

3´ 5´
5´ 3´

3´ 5´
5´ 3´
95°C for 30 sec

3´ 5´
5´ 3´
56°C for 30 sec

3´ 5´
5´ 3´

3´ 5´

 By repeating these cycles again and again,
enormous amplification of the target sequence
of DNA can be achieved
 After twenty cycles, nearly one million copies
are formed
 After thirty cycles, nearly one billion copies are
formed
 This can be done automatically in instruments
known as thermocyclers

Strand separation
(95°C for 30 sec)
Primer
binding
( 55°C for 30 sec)
Replication
(75°C for 2-5 min)
Strand
separation
(95°C for 30 sec)

Temperature cycles in thermocycler

TECHNIQUES FOR IDENTIFICATION OF DNA
AND RNA
 Techniques for identification of DNA fragments,
recombinant DNA, RNA and proteins are often
required in recombinant DNA technology
 Generally, complementary DNA (cDNA) probes
are prepared and used for identification of DNA
and RNA
 cDNA probes hybridise with complementary
DNA or RNA

 Proteins can be identified by using specific
antibodies as probes
 DNA, RNA or proteins are separated by
electrophoresis on agarose gel (for high
molecular weight compounds) or
polyacrylamide gel (for low molecular weight
compounds), and transferred to a nitrocellulose
sheet
 A probe labelled with 32P is added which binds
to the band of interest

 The band becomes visible on exposing the sheet
to an x-ray film (autoradiography)
 A technique for identification of a specific DNA
was devised by Southern (1975)
 This technique is known as Southern blot
transfer or Southern blotting
 SNOW
 DROP

 A similar technique for identification of RNA
was later devised by Alwine et al (1977), and
was jokingly named as Northern blot transfer
or Northern blotting (use Diazobenzyloxymethyl
-DBM in place of nylon membrane)
 In this technique, a cDNA–RNA hybrid is
formed
 A similar technique for identification of
proteins with the help of labelled antibody
probes was devised later, and was named
Western blot transfer or Western blotting

TECHNIQUES FOR DETERMINATION OF
BASE SEQUENCE OF DNA
 The base sequence of DNA can be
determined by :
• The chemical method of Maxam-Gilbert
• The enzymatic dideoxy method of Sanger

Maxam-Gilbert method
 This method is based on chemical cleavage of
DNA at selective sites
 The DNA to be analysed is labelled at its 3’-end
with a nucleotide having 32P
 Reagents are available which can selectively
destroy and remove one particular base from
DNA

 Four sets of labelled DNA are treated with four
different reagents which destroy and remove
adenine, guanine, cytosine and thymine
respectively
 Conditions are so chosen that only one base is
removed per DNA strand

 When several strands of DNA are treated with
a particular reagent, the given base is removed
randomly from all the possible sites where it was
present
 Chain breaks are, then, induced at the “base-
less” sites

 The fragments produced in the four tubes are
separated by electrophoresis on
polyacrylamide gel in four parallel lanes
 Locations of 32P-labelled fragments are
identified by autoradiography
 The smallest fragment moves the farthest from
the point of application, and the largest
fragment moves the least

 The relative sizes of the fragments indicate the
distance between the 32P-label and the
destroyed base
 By arranging the fragments in the four lanes in
the decreasing order of size, the sequence of
bases from 5’–end to 3’-end can be deduced

5’ ATCGATCG 3’
5’ ATCGATCGC* 3’
Addition of radiolabelled
C* at 3’- end
Removal
of A
Cleavage at
“baseless” site
Electrophoresis and
autoradiography
5’–TCGATCGC*3’
and
5’ATCG–TCGC*3’
5’ATC–ATCGC*3’
and
5’ATCGATC–C*3’
5’AT–GATCGC*3’
and
5’ATCGAT–GC*3’
5’A–CGATCGC*3’
and
5’ATCGA–CGC*3’
Removal
of G
Removal
of C
Removal
of T

TCGATCGC*
and
ATCG+TCGC*
ATC+ATCGC*3
and
ATCGATC+C*
AT+GATCGC*
and
ATCGAT+GC*
A+CGATCGC*
and
ATCGA+CGC*
8
7
6
5
4
3
2
1
A 5´
T
C
G
A
T
C
G 3´
A
Lane
G
Lane
C
Lane
T
Lane

  


   




Sanger’s dideoxy method
 This method using dideoxynucleotides was
devised by Frederick Sanger
 Sanger had earlier devised a method for protein
sequencing

Frederick Sanger

The 3’-OH group can form a
phosphodiester bond with the
next nucleotide
There is no 3’-OH group to form
a phosphodiester bond with the
next nucleotide
H
H
H
OH
H
H
CH2
Base
O
P ~ P ~ P — O
H
H
H
H
H
CH2
Base
O
P ~ P ~ P — O
H
2’-Deoxyribonucleoside triphosphate 2’,3’-Dideoxyribonucleoside triphosphate
O
O

 The DNA strand to be sequenced is used as a
template for replication
 Four tubes are set up in each of which, the
template strand, 32P-labelled primer, dATP,
dGTP, dCTP, dTTP and DNA polymerase are
added
 In each tube, one dideoxynucleotide (ddATP,
ddGTP, ddCTP or ddTTP) is added

 The dideoxynucleotide competes with the normal
nucleotide
 If a normal nucleotide enters the growing chain,
replication continues
 If a dideoxynucleotide enters the growing chain,
replication stops

 The relative concentrations of the
dideoxynucleotide and its normal counterpart are
such that the dideoxy analogue enters
randomly at different sites in different cycles
of replication
 After several cycles of replication, chains of
different lengths, each ending with a
dideoxynucleotide, are formed
 The chains formed in the four tubes are
separated by electrophoresis on
polyacrylamide gel in four parallel lanes, and are
autoradiographed

 The pattern of bands on the autoradiogram
gives the sequence of bases which is
complementary to the template strand
 Sanger’s method has also been automated
which has made DNA sequencing much faster

Electrophoresis and
autoradiography
8
7
6
5
4
3
2
1
G
C
T
A
G
C
T
A
A
Lane
G
Lane
C
Lane
T
Lane
5’–ATCGATCG
3’ TAGCTAGC
–
   
5’ ATCGATCG
3’ TAGCTAGC
–
–
5’ ATCGATCG
3’ TAGCTAGC
–
–
5’ ATCGATCG
3’ TAGCTAGC
–
–
5’ ATCGATCG
– 5’ ATCGATCG
– 5’ ATCGATCG
– 5’ ATCGATCG
–
3’ TAGCTAGC
– 3’ TAGCTAGC
– 3’ TAGCTAGC
– 3’ TAGCTAGC
–
   
3’ TAGCTAGC
5’
– 3’ TAGCTAGC
5’
– 3’ TAGCTAGC
5’
– 3’ TAGCTAGC
5’
–

dATP,
dGTP,
dCTP,
dTTP,
ddATP*,
DNA
poly-
merase

dATP,
dGTP,
dCTP,
dTTP,
ddGTP*,
DNA
poly-
merase

dATP,
dGTP,
dCTP,
dTTP,
ddCTP*,
DNA
poly-
merase

dATP,
dGTP,
dCTP,
dTTP,
ddTTP*,
DNA
poly-
merase
3’ TAGCTAGC
5’ A*

– 3’ TAGCTAGC
5’ ATCG*

– 3’–TAGCTAGC
5’ ATC*

3’ TAGCTAGC
5’ AT*

–
+ + + +
3’ TAGCTAGC
5’ ATCGA*

– 3’ TAGCTAGC
5’ ATCGATCG*

– 3’ TAGCTAGC
5’ ATCGATC*

– 3’ TAGCTAGC
5’ ATCGAT*

–
Tube 1 Tube 2 Tube 3 Tube 4
3’
5’






APPLICATIONS OF RECOMBINANT DNA
TECHNOLOGY
 Recombinant DNA technology finds applications
in all branches of life sciences and industry
 Advent of recombinant DNA technology has led
to spectacular advances in medical science

 Some of the applications of recombinant DNA
technology in medicine are in :
1. Mapping of genomes
2. Production of proteins
3. Diagnosis of genetic diseases
4. Medico-legal applications
5. Gene therapy

Mapping of Genomes
 Mapping of genome means determining the
base sequence of entire DNA of an organism
 Since the size of DNA is very big, it has to be
broken up into small fragments so that the
sequencing becomes easier
 Fragments of DNA obtained from a genome are
amplified and maintained in what is known as a
library

Genomic library
 The total DNA in a cell is hydrolysed by restriction
enzymes to yield fragments of 15-25 kb
 The fragments are separated by electrophoresis
 Each fragment is ligated to a bacteriophage
vector e.g. phase lgt 10 or lgt 11

 The vector is allowed to infect E. coli
 Multiple copies of different DNA fragments
will be formed in different E. coli cells
 Each clone of the vector would contain one DNA
fragment
 A collection of such clones is known as genomic
library of the organism

cDNA library
 The genomic library is made from total DNA
which includes coding sequences as well as
non-coding sequences
 Non-coding sequences are present in genes in
the form of introns and as long stretches in
between the genes
 If a library of only structural genes is desired,
one can prepare a cDNA (complementary
DNA) library

 cDNA library is prepared from mRNA templates
 Total RNA is isolated from a cell or a tissue
 It is passed through a column having poly-T
oligonucleotides fixed to an inert gel
 The poly-T oligonucleotides bind the poly-A tails
of mRNA molecules while the other types of
RNA pass through the column

 The mRNA molecules are later eluted and used
as templates for synthesizing a cDNA strand by
reverse transcriptase
 The RNA template is hydrolysed by
ribonuclease H
 The cDNA strand is than used as a template to
synthesise the second strand of DNA by DNA
polymerase

 Such double-stranded DNA molecules are ligated
to a vector, e.g. a plasmid, and amplified and
maintained in E. coli cells
 The entire collection of these vectors constitutes
a cDNA library

Techniques to locate a desired DNA insert in the vector
(i) If the base sequence of the desired DNA insert is
known, a radio-labelled cDNA probe can be
used to identify the particular vector by
autoradiography
(ii) If the base sequence is not known, we can look
for the protein encoded by the DNA insert with
the help of an antibody probe or by assaying its
function

Production of Proteins
 Proteins of diagnostic, therapeutic, nutritional or
industrial importance can be produced in large
quantities by recombinant DNA technology
 Human insulin was the first protein to be
synthesised in E.coli by this technology

 Human growth hormone, interferon, tissue
plasminogen activator, Factor VIII,
erythropoietin etc are being synthesised by
this technology
 Bovine growth hormone and subtilisin, a
proteolytic enzyme used in detergents, are
being produced by recombinant DNA
technology
 Human albumin and enkephalin genes have
been transferred into plants

 Recombinant vaccines and antibodies are being
synthesised for laboratory and clinical use
 By site-directed mutagenesis, specific
alterations can be made in the amino acid
sequence of a protein
 The vectors used to introduce genes into cells
for the purpose of protein synthesis are known
as expression vectors which include
plasmids, phages, baculovirus, vaccinia virus
etc

Synthesis of recombinant human insulin
 Human insulin gene is constructed by reverse
transcription of mRNA for insulin
 Natural insulin gene is not used as it contains
introns which cannot be removed by bacteria
 The constructed insulin gene is introduced into
E.coli with the help of a plasmid vector
 E.coli multiplies and synthesises vast quantities
of insulin which can be extracted and purified

 A vaccine against hepatitis B has been
prepared in yeast
 Vaccines against a variety of infectious diseases
are traditionally prepared from killed or live
attenuated micro-organisms
 In either case, there is some risk of infection if
some potent, infectious micro-organisms
remain in the vaccine

 This risk can be eliminated if the vaccine
contains only the antigenic protein and not the
DNA or RNA of the micro-organism
 Such a vaccine is known as a subunit vaccine
 Hepatitis B virus has a surface antigen (HbsAg)
in its coat which is antigenic but not infectious

 The gene for HbsAg is isolated from the viral
genome, and is ligated to yeast plasmid
 The plasmid is introduced into yeast which
multiplies and synthesises large quantities of
HbsAg
 HbsAg is isolated and is used as a vaccine

Diagnosis of Genetic Diseases
 Genetic diseases result from mutations e.g.
substitution, insertion or deletion
 Mutations can occur in non-coding as well as
coding regions of DNA
 Mutations in non-coding regions do not impair
function but result in polymorphism

 1. Restriction fragment length
polymorphisms
(RFLPs, pronounced as rif-lips).
 2. Minisatellites or variable number tandem
repeats
(VNTRs, pronounced as vinters).
 3. Microsatellites or simple tandem repeats
(SIRs).
 4. Single nucleotide polymorphisms
(SNPs, pronounced as snips).

 If DNA of normal individuals is treated with a
restriction enzyme, restriction fragments of
varying length are formed depending on
the number of restriction sites in the DNA
 The restriction pattern is inherited, and results
in restriction fragment length polymorphism
(RFLP)
 A mutation in coding region can create a new
restriction site or can obliterate a restriction
site, thus, changing the number and length of
restriction fragments

 For example, three restriction sites for Mst II
are normally present in the b-globin gene
 Therefore, Mst II produces two restriction
fragments
 In sickle cell anaemia, the single base
substitution obliterates one of the restriction
sites for Mst II with the result that only one,
larger fragment will be produced by Mst II

Normal b- globin gene
1.15 kb 0.2 kb
b- Globin gene (Hb S)
1.35 kb
Broken arrows show restriction sites for Mst II

 Thus, the change in RFLP pattern can be used
to diagnose the disease
 Several genetic diseases can be diagnosed
pre-natally by obtaining DNA from amniotic
fluid, amplifying it by PCR and studying its
RFLP pattern

 It is also possible to diagnose infectious
diseases by detecting specific bacterial or
viral genes in a biological sample with the
help of complementary probes
 Even if the number of micro-organisms in the
sample is very small, PCR can be used to
amplify the DNA

Applications of RFLPs
 Sickle-cell anemia - (chromosome 11 )
 Cystic fibrosis - (chromosome 7)
 Huntington's disease - (chromosome 4)
 Retinoblastoma - (chromosome 13)
 Alzheimer 's disease - ( chromosome 21)

Medico-legal Applications
 Genomes of higher organisms, including
human beings, contain some short repetitive
sequences in the non-coding regions which
are scattered throughout the genome
 Such sequences are called tandem repeats
 The number of repeats varies in different
persons
 This phenomenon is called variable number of
tandem repeats (VNTR)

 VNTR pattern is inherited from parents in a
Mendelian fashion
 VNTR pattern is unique for each individual,
and there are striking resemblances between
close blood relations e.g. parents and
offsprings
 Restriction sites for various restriction
enzymes are present on the flanks of many
tandem repeats
 cDNA probes have been developed for several
tandem repeats

 When the DNA is treated with suitable
restriction enzymes, a number of fragments
having tandem repeats will be formed
 The relative lengths of the fragments will
depend upon the number of tandem repeats
 The fragments can be separated by
electrophoresis and, using suitable cDNA
probes, their positions can be seen on
autoradiograms

 If three or four different repetitive sequences
are identified by cDNA probes, the
autoradiographic pattern becomes
unique for each individual
 The pattern is so unique that it is called as the
DNA finger-print of the individual
 DNA finger printing has tremendous
applications in forensic medicine

 If a criminal has left behind some biological
material, e.g. hair, blood stain, semen stain
etc, at the scene of crime, DNA can be
extracted, amplified by PCR and its VNTR
pattern can be established
 This can be compared with the VNTR pattern
of suspects, and the real culprit can be
identified
 In cases of disputed parenthood, VNTR
pattern of the child can be compared with
that of suspected father/mother, and
paternity/maternity can be established

Gene Therapy
 Treatment of genetic diseases by introducing
normal genes into the DNA of patients was
an impossible task before the advent of
recombinant DNA technology
 With the development of techniques for DNA
sequencing, cloning of genes and availability
of expression vectors, gene therapy has
now become a practical reality

 Gene therapy may be tried in an embryo if pre-
natal diagnosis can be made or in a patient in
whom a genetic disease has been diagnosed
 Some successful experiments of both types
have been done in animals in diseases like
cystic fibrosis, Lesch- Nyhan syndrome,
Duchenne muscular dystrophy,
thalassaemia etc

 Initial trials of gene therapy were done in
transgenic animals and knock out animals
 Transgenic animals are prepared by micro-
injecting a foreign gene into a fertilised ovum
 The gene gets stably incorporated in the genome
of the animal, and is transmitted to future
generations as well

 Knock out animals are prepared by deleting a
particular gene from a fertilised ovum
 In this way, a particular genetic disease can be
produced in the knock out animal

 Gene therapy in human embryos poses some
ethical problems
 Introduction of foreign DNA can cause
unforeseen changes in host DNA which would
be stably incorporated in the genome of germ
cells, and would be transmitted to the future
offsprings
 Genetic manipulations in somatic cells do not
pose this problem as the change would affect
only one individual

 However, targeting the foreign gene to a specific
destination, e.g. brain, liver, pancreas etc, is still
a problem
 Gene therapy of blood cells and bone marrow
cells doesn’t pose this problem as the gene-
treated cells can be easily introduced in
circulation or bone marrow

 The first clinical trial of gene therapy in human
beings was undertaken in 1990 in USA in a
disease, severe combined immunodeficiency
disease (SCID) caused by mutations in
adenosine deaminase (ADA) gene
 ADA deficiency cripples the immune system
 The affected children are extremely prone to
infections, and rarely survive beyond early
childhood without specialised care

 Gene therapy was started in two children
suffering from SCID
 T lymphocytes were isolated from their blood
 Normal ADA gene was introduced in these cells
with the help of a disabled retroviral vector,
and the cells were put back into circulation

 Since the life-span of these cells is limited, the
treatment was repeated every month
 The children showed a significant increase in
their T cell count and increased ADA levels in T
cells
 Their immune system showed significant
improvement, and they were able to fight
infections

 The treatment was stopped after two years but
clinical improvement persisted even after
cessation of gene therapy
 Since then, several more children with SCID
have been successfully treated by gene therapy

 The success of gene therapy in SCID has
opened new vistas for the treatment of genetic
and even non-genetic diseases
 Advanced clinical trials are in progress in human
beings for gene therapy of ischaemic
vascular diseases
 The introduction of the gene for vascular
endothelial growth factor (VEGF) has given
promising results so far

 With refinements in technology and further
research, gene therapy is expected to be used
successfully in many human diseases in future

• Disease
• Severe combined
immunodeficiency(SCID)
• Cystic fibrosis
• Familial hypercholesterolemia
• Emphysema
• Hemophilia B
• Thalassemia
• Sickle-cell anemia
• Lesch - Nyhan syndrome
• Gaucher’s disease
• Peripheral artery disease
• Gene therapy
• Adenosine deaminase (ADA)
• Cystic fibrosis transmembrane
regulator (CFTR).
• LDL Receptor
• α1-Antitrypsin
• Factor lX
• α or β-Globin
• β -Globin
• Hypoxanthine-guanine
phosphoribosyle
transferase (HGPRTase).
• β-Glucocerebrosidase
• Vascular endothelial growth
factor (VEGF)

• Fanconi anemia
• Melanoma
• Melanoma, renal cancer
• Glioblastom(barain tumor),
AIDS, ovarian cancer
• Head and neck cancer
• Breast cancer
• AIDS
• Colorectal cancer,
melonema, renal cancer
• Duchenne muscular
dystrophy
• Short stature
• Diabetes
• Phenylketonuria
• Citrullinemia
• Fanconi anemia C
• Tumor necrosis factor (TNF-α)
• Interleukin -2 (lL-2 )
• Thymidin kinase (herpes
simplex virus)
• p53
• Multidrug resistance
• rev and env
• Histocompatability locus
antigen-B7 ( HLA-B7)
• Dystrophin
• Growth hormone
• Glucose transporter (GLUT-2),
glucokinase
• Phenyl alanine hydroxylase
• Arginosuccinate synthetase

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