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Recombinant DNA Technology 22.ppt
1. 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
2. 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
3. Recombinant DNA technology has proved to be
of immense value in :
• Medicine
• Agriculture
• Animal husbandry
• Industry
4.
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
7. 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
8. 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
9. 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
10. 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
11. 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)
12. 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
13. 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
14. 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
15. 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
16. 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
+
+
+
17. 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
18.
19. 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
+ + +
– – – – – – – –
– – – – – – – –
20. 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
21.
22. CLONING OF RECOMBINANT DNA
Cloning of DNA was pioneered by Herbert Boyer
and Stanley Cohen
Herbert Boyer Stanley Cohen
23. 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
24. 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
25. 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
26. 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
27.
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
28. 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
29. 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
30. 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
31. 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
34. The virus infects a bacterial cell by injecting its DNA into the
bacterial cell – Phase ƛ and Phase M13
35.
36. After entering the bacterial cell, the viral DNA
may enter one of the two alternate pathways:
1. Lysogenic pathway
2. Lytic pathway
37. 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
38. Lytic pathway
In the lytic pathway, the viral DNA remains
separate
It replicates independently
39. 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
40. 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
41. 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
42. 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
43. POLYMERASE CHAIN REACTION
Polymerase chain reaction (PCR) is a technique
for amplification of DNA devised by Mullis(1984)
Kary Mullis
44. 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
45. 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
46. 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
47. 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
48. 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´
49. 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
50. 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
51. 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
52. 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
53. 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
55. 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
56. 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
57. 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
58. 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
59. 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
60. 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
61. 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
62. 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
63. Sanger’s dideoxy method
This method using dideoxynucleotides was
devised by Frederick Sanger
Sanger had earlier devised a method for protein
sequencing
Frederick Sanger
64. 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
65. 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
66. 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
67. 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
68. 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
69. 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’
70. 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
71. 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
72. 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
73. 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
74. 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
75. 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
76. 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
77. 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
78. 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
79. 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
80. 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
81. 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
82. 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
83. 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
84.
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
85. 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
86. 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
87. 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
88. 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
89. 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).
90. 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
91.
92. 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
93. 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
94. 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
95. 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
97. 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)
98. 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
99. 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
100. 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
101. 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
102. 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
103. 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
104. 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
105. 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
106. 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
107. 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
108. 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
109. 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
110. 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
111. 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
112. 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
113. With refinements in technology and further
research, gene therapy is expected to be used
successfully in many human diseases in future