Methods used to prepare and amplify recombinant DNA; identification of specific DNA, RNA and proteins, DNA sequencing

1. Recombinant DNA Technology:
Tools and Techniques
R. C. Gupta
M.D. (Biochemistry)
Jaipur, India

2. Recombinant DNA technology is used to
produce recombinant DNA
Recombinant DNA is made by combining
different fragments of DNA
Usually, the DNA fragments are derived
from different sources

3. Recombinant DNA, having unrelated
genes, is also known as chimeric DNA
Chimera is a mythological
creature having the head of a
lion, the trunk of a goat and
the tail of a serpent

4. Recombinant DNA technology came into
existence in the 1970s
It has led to revolutionary changes not
only in biochemistry but in all life sciences

5. Recombinant DNA
technology has proved to
be of immense value in:
Medical science
Agriculture
Animal husbandry
Industry

6. Opened the doors of
recombinant DNA research
The most important tools of
recombinant DNA technology
Restriction endonucleases
Discovered by Arber, Smith and
Nathans in the early1970s
Arber
Smith
Nathans

7. Restriction endonucleases (enzymes)
are found in bacteria
They protect the bacteria against viral
infections
When a virus infects a bacterial cell,
restriction enzymes split the viral DNA
Thus, the virus is destroyed

8. Hundreds of restriction enzymes
have been discovered so far
They are named after the bacterium
in which they are found e.g. Hin I,
Hae III, Eco RI

9. The three-letter abbreviation is
derived from the name of the
bacterium e.g.
Hin from Haemophilus influenzae
Hae from Haemophilus aegyptius
Eco from 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 enzymes from
the same bacterium in the order of
their discovery

11. Each restriction enzyme:
Recognizes a specific base
sequence in double-stranded DNA
Splits the DNA at this site

12. The base sequence recognized
by a restriction enzyme:
Is 4-8 base pairs long
Is palindromic

13. A palindrome is a word or a sentence
which reads the same from left to right
and from right to left e.g. DAD,
MADAM, RADAR etc

14. In DNA, the base sequences are read in
5’ → 3’ direction
If a sequence reads the same on both the
strands in 5’ → 3’ direction, it is known as
a palindromic sequence
5’ GGCC 3’ 5’ TTTAAA 3’
3’ CCGG 5’ 3’ AAATTT 5’

15. Restriction enzymes:
Split both the strands of DNA
Produce blunt or sticky ends
Blunt ends Sticky ends

16. Blunt ends are also known as even or
non-overlapping ends
Sticky ends are also known as cohesive
or overlapping ends

17. 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

18. Sticky ends are more useful in
recombinant DNA technology
They can be easily ligated to the
complementary sticky ends of another
fragment of DNA by DNA ligase
GGCCTCAAT
AGTATCCGG

19. Two complementary sticky ends joined
Restriction enzyme
DNA ligase
Sticky ends of two fragments joined
Fragment having
sticky end
Fragment having
sticky end
Another DNA cut by
the same enzyme
The enzyme cuts both the
strands at its restriction site
A
T T A A
A T T

20. The site recognized by a restriction enzyme
is known as its restriction site
DNA fragments produced by restriction
enzymes are known as restriction fragments
Every DNA has got a number of restriction
sites for a number of restriction enzymes

21. 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
4
4
4 +
+
+
5
6
6
6
5
5
Restriction sites of some enzymes

22. Recombinant DNA:
Can be formed by joining restriction
fragments obtained from different
sources
Will have a base sequence different
from that of the original DNA

23. A foreign gene can be inserted into DNA
by this method
The gene is clipped out using a restriction
enzyme
It is ligated to another DNA using DNA
ligase

24. 5’ – – GGATCC – – 3’
3’ – – CCTAGG – – 5’
Foreign gene
DNA having Bam HI
restriction site
Foreign gene having Bam HI restriction
site on either side
6
Bam HI
6
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
+ + +
6
5’ – – GGATCC – – – – – – – – GGATCC – – 3’
3’ – – CCTAGG – – – – – – – – CCTAGG – – 5’

25. Amplification
Amplification is often required to prepare
multiple copies of the recombinant DNA
Amplification can be done:
Either by cloning the DNA
Or by polymerase chain reaction

26. Herbert Boyer Stanley Cohen
Cloning of recombinant DNA
Cloningof DNA was pioneered by Herbert Boyer and
StanleyCohen

27. DNA can be cloned in living cells
A vector is required to transfer
recombinant DNA into the cells
A clone is a population of identical
organisms or cells or molecules
derived from the same source

28. Some useful cloning vectors are:
Plasmids
Bacteriophages
Cosmids
Bacterial artificial chromosomes
Yeast artificial chromosomes

29. Plasmids are small circular double-
stranded DNA molecules
They are present in prokaryotes in
addition to the chromosomal DNA
They may replicate independently of the
chromosomal DNA
Plasmids

30. Plasmids contain one or more antibiotic-
resistance genes
These genes provide antibiotic-resistance
to the bacteria
Plasmids can be transferred from one
bacterium to another
Plasmids can accept foreign DNA frag-
ments up to 10 kb in size

31. Therefore, DNA fragments up to
10 kb (kilo bases) in size can be
easily inserted into plasmids, and
cloned in bacteria e.g. E.coli

32. Nick the plasmid by a restriction enzyme
to generate sticky ends
Ligate the desired DNA fragment having
complementary sticky ends to the plasmid
by DNA ligase
Introduce the plasmid into a bacterial cell
Technique

34. Multiplication of the bacterial cell
and independent replication of the
plasmid produce multiple copies of
the foreign DNA within a short time

35. pSC 101 is a plasmid present in E. coli
This was the first vector used for cloning
pSC 101 has a single restriction site, a
single antibiotic-resistance gene and it
replicates poorly

36. An ideal vector should replicate rapidly,
should have several restriction sites for
different restriction enzymes and more
than one antibiotic-resistance genes

37. Some plasmids having these features
have been constructed in the laboratory
Examples are pBR 322, pBR328, pUC18
etc

38. Plasmids can be introduced into bacteria
by exposing them to high concentration of
divalent cations
This increases the permeability of
bacterial cell membrane so as to allow
plasmids to enter the bacterial cells

39. However, plasmids may not enter all the
bacterial cells
Some plasmids might not have taken up
the foreign DNA
We have to select the bacterial cells
having recombinant plasmids

40. Antibiotic-resistance genes are useful in
selection of recombinant plasmids
For example, plasmid pBR322 has got
ampicillin- and tetracycline-resistance genes
Restriction sites are present in the middle of
these genes

41. Ampicillin-resistance gene
Tetracycline-resistance gene
pBR322

42. A foreign gene/DNA fragment can be inserted
in the middle of an antibiotic-resistance gene
Ampicillin-resistance gene
Tetracycline-resistance gene (disrupted)
Foreign DNA

43. Bacteria having recombinant plasmid
would lose resistance to tetracycline
But they would be resistant to ampicillin

44. We first grow the bacteria on an agar
plate containing ampicillin
The bacterial cells which have taken up
and those which have not taken up the
recombinant plasmid will grow

45. We partially transfer the bacterial colonies
on a replica plate containing tetracycline
Cells having the recombinant plasmid will
be destroyed
Thus, the colonies on the original plate
having the recombinant plasmid can be
identified

46. Agar plate
containing
ampicillin
Replica plate
containing
tetracycline
Colonies having
recombinant
plasmid

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

48. The virus infects a bacterial cell by
injecting its DNA into the bacterial cell

49. Landing Pinning
DNA injectionTail contraction
and penetration

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

51. 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
Lysogenic pathway

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

53. The proteins encoded by the viral DNA
are synthesized by the bacterial cell
Each DNA molecule is packaged into a
protein coat forming a new virus particle
When the number of viruses becomes
very large, the bacterial cell ruptures

54. The provirus can break out of the
bacterial DNA and enter the lytic pathway
if the bacterial cell is exposed to some
DNA damaging agent e.g. ultra-violet light

55. Bacteriophages can be used as vectors
for cloning
A portion of viral DNA, not essential for its
replication and packaging, is clipped out
by restriction enzymes
The foreign DNA to be cloned is inserted
in the viral DNA

56. The virus infects a bacterial cell
It multiplies inside the bacterial cell
A large number of copies of foreign DNA
are formed in the bacterial cell

57. DNA is packaged
into protein coat
Phage DNA
Restriction enzyme
Foreign DNA
DNA ligase
Recombinant phage DNA
E. Coli cell Virus attaches to
E. Coli cell; injects
its DNA into the cell
Infected
E. Coli cell
Viral DNA
replicates in
the E. coli cell
Virus
Restriction fragments

58. Lambda phage and M13 phage are
commonly used as cloning vectors
DNA fragments up to 20 kb in size can be
inserted in phage vectors, and can be
cloned in E.coli
Moreover, it is easier to infect E. coli with
a phage than with a plasmid

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

60. 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

61. Origin of
replication
Ampicillin-resistance
gene
Restriction sites
Cos siteCosmid

62. BAC is a genetically engineered vector
BAC is constructed from a fertility plasmid
(F factor)
Bacterial artificial chromosome (BAC)

63. BAC possesses chloramphenicol-resistance
gene
This gene helps in selecting the cells having
recombinant DNA
A polylinker contains several restriction sites
for inserting foreign DNA

64. Origin of
replication
Chloramphenicol-
resistance gene
Restriction sites
BAC

65. BAC can be used to clone DNA in
bacterial cells e.g. E. coli
BAC can accept foreign DNA of 100-300
kb
BAC is nicked with a restriction enzyme
and foreign DNA is inserted in it

66. The BAC is introduced into a bacterial cell
It acts as an extra chromosome in the
bacterial cell
As the bacterial cell divides, the BAC is
also replicated

67. Multiple copies of BAC are formed in the
bacterial cells
The BAC can then be isolated
BACs are commonly used to clone DNA
for determining its base sequence

68. YACs are formed by adding yeast telo-
meres to a centromere-containing plasmid
It has got an autonomous replicating
sequence necessary for replication
It has also got some restriction sites and
markers
Yeast artificial chromosome (YAC)

69. Eco RI
restriction site
BamHI
restriction site
Marker
TelomereTelomere
Marker
Centromere
Autonomous
replicating sequence
Ampicillin-
resistance gene
Yeast artificial chromosome

70. YAC can accept large fragments of
foreign DNA
Foreign DNA up to 3,000 kb in size can
be inserted in YAC, and cloned in yeast
cells

71. Polymerase chain reaction (PCR)
Polymerase chain reaction is
a technique for rapid ampli-
fication of DNA devised by
Kary Mullis in the 1980s

72. PCR is far quicker, easier and cheaper
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

73. The DNA to be amplified is replicated by
DNA polymerase of Thermus aquaticus
(Taq) in PCR
Thermus aquatics is a bacterium found in
hot water springs

74. T. aquaticus is used to high temperatures
Its enzymes are not denatured even at
95°C
Optimum temperature of its enzymes,
including DNA polymerase, is 72°C

75. In PCR, DNA has to be heated to 94-95°C
for separation of strands
Since DNA polymerase of T. aquaticus is
not destroyed at this temperature, it is an
ideal enzyme for PCR
DNA polymerase of T. aquaticus is usually
called Taq polymerase

76. The reaction mixture for PCR contains:
• The DNA to be amplified
• Taq polymerase
• A large quantity of primers
• Deoxyribonucleotides (dATP, dGTP,
dCTP & dTTP)
• Buffer with Mg++ and K+

77. Primers have to be added to the reaction
mixture as they are required to initiate
each cycle of replication
To prepare primers, we must know short
flanking sequences on either side of the
target DNA sequence

78. Amplification occurs in three steps:
Strand separation
Primer binding
Primer extension (addition of
deoxyribonucleotides to primer)

79. The temperature is raised to
95°C to separate the DNA
strands
First
step
The temperature is lowered
to 56°C for binding of
primers to DNA strands
Second
step
The temperature is raised to
72°C for Taq polymerase to
replicate the strands
Third
step

80. Target sequence of DNA
to be amplified
The two strands separate
Primers hybridize with
template strands
Taq polymerase replicates
both the strands. Two copies
of target DNA are formed
Target
sequence
95°C for 30 sec
56°C for 30 sec
72°C for 2 to 5 min
3’
´
5’
5’
´
3’
´
5’ 3’
´
3’ 5’
´
5’
5’
3’
3’

81. 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
72°C for 2-5 min
´
95°C for 30 sec
56°C for 30 sec
3’ 5’
5’ 3’
5’ 3’
3’ 5’
3’5’
3’ 5’
3’5’
3’ 5’
Second cycle begins

82. By repeating the cycles again and again,
enormous amplification can be achieved
After twenty cycles, nearly a million copies
of target sequence of DNA are formed
After thirty cycles, nearly a billion copies
are formed

83. After preparing the reaction mixture, the
only thing to be done to repeat the cycles
is to change the temperature cyclically
This can be done auto-
matically in instruments known
as thermocyclers

84. Strand separation
(95°C for 30 sec)
Primer
binding
(56°C for
30 sec)
Replication
(72°C for
2-5 min)
Strand
separation
(95°C for
30 sec)
Temperature cycles in thermocycler

85. Several new variants of the
classical PCR have been devised:
Nested PCR
Multiplex PCR
Reverse transcriptase-PCR
Quantitative PCR

86. Amplification is done twice using two
different sets of primers
One pair of primers is used first to
produce a DNA product
This may contain unintended DNA
sequences besides the intended target
Nested PCR

87. The product of first PCR is then used as
target sequence in the second PCR
Different primers are used that bind
downstream from the primers used first
Nested PCR increases the specificity of
DNA amplification

88. Nested PCR
First primer
|
Second primer
|

89. Multiple sets of primers are used in the
same run
Different target sequences are amplified
in a single run
Multiplex PCR

90. Multiplex PCR

91. A DNA complementary to an RNA is
prepared by reverse transcription
This is amplified by PCR
This DNA can be used to determine the
base sequence of RNA
Reverse transcriptase-PCR

92. Fluorescent dyes are used to measure the
amount of the amplified DNA in real time
Quantitative PCR is also known as real
time PCR
Quantitative PCR

93. The initial PCR methods were able to
amplify DNA of the size of 3 kb only
With refinements in methodology, the size
has increased to10 kb now

94. Applications of PCR
• Study of genes
• Genome mapping
• Diagnosis of genetic diseases
• Diagnosis of infectious diseases
• DNA finger printing

95. Techniques for identification of
DNA and RNA
In recombinant DNA technology, we
often require techniques to identify a
specific:
DNA RNA Protein

96. Complementary DNA (cDNA) probes are
used for identification of DNA and RNA
The cDNA probe hybridizes with comple-
mentary DNA or RNA strand
Antibodies can be used as probes to
identify specific proteins

97. A technique for identification of a specific
DNA was devised by Southern in1975
This technique is known as Southern blot
transfer or Southern blotting

98. DNA is hydrolysed by a restriction enzyme
The DNA fragments are separated by gel
electrophoresis
Agarose gel is used for high molecular
weight compounds
Polyacrylamide gel is used for low
molecular weight compounds
Southern blotting

99. Samples
DNA ladder
Gel
Gel
Gel
Nitrocellulose sheet
Nitrocellulose
sheet
Sponge
Buffer
Paper towels
The fragments are transferred to a nitrocellulose sheet

100. A probe labeled with 32P is added which
binds to the band of interest
Unbound probe molecules are washed off
The sheet is exposed to an x-ray film
The band of interest becomes visible
(called autoradiography)

101. Southern blot transfer
Unbound probe washed off and the
sheet exposed to x-ray film
32P-Labelled
probe added
Nitrocellulose
sheet overlaidElectrophoresis
Band having
the probe
becomes visible
Probe find the
complementary
fragment and
binds to it
Fragments are
transferred onto
nitrocellulose sheet
Fragments are
separated and
melted (denatured)
DNA fragments
produced by restriction
enzymes are put on gel

102. A similar technique for identification of
RNA was devised in 1977 by Alwine
This was jokingly named as northern blot
transfer or northern blotting
In this technique, a cDNA-RNA hybrid is
formed
Northern blotting

103. Northern blot transfer

104. A similar technique for identification of
proteins was devised later by Burnette
The protein of interest is identified with the
help of a labelled antibody probe
This technique was named as western blot
transfer or western blotting
Western blotting

105. Western blot transfer

107. Techniques for determining base
sequence of DNA
The base sequence of DNA
can be determined by:
Chemical method of Maxam-Gilbert
Enzymatic dideoxy method of Sanger

108. Maxam-Gilbert method is also known as
selective chemical cleavage method
It is based on chemical cleavage of DNA
at selective sites
Maxam-Gilbert method

109. The DNA to be sequenced is labeled at its
3’-end with a nucleotide having 32P
Reagents that destroy/remove one specific
base are used to remove bases from DNA

110. Four sets of 32P labeled DNA are treated
with four different reagents
These reagents remove adenine, guanine,
cytosine and thymine respectively
Conditions are so chosen that only one
base is removed per DNA strand

111. Several strands of DNA are treated with
each 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

112. The fragments produced in the four tubes
are separated by electrophoresis on poly-
acrylamide gel in four parallel lanes
Locations of 32P-labeled fragments are
identified by autoradiography

113. The smallest fragment moves the
farthest from the point of application
The largest fragment moves the least
The other fragments are in between,
depending on their size

114. Relative sizes of the fragments indicate
the distance between the 32P-label (3’-end)
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

115. 5’ ATCGATCG3’
5’ ATCGATCGC*3’
Addition of radiolabeled C* at 3’-end
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’
TCGATCGC*
and
ATCG+TCGC*
ATC+ATCGC*3
and
ATCGATC+C*
AT+GATCGC*
and
ATCGAT+GC*
A+CGATCGC*
and
ATCGA+CGC*
1
2
3
4
5
6
7
8
5´
3´A
Lane
G
Lane
C
Lane
T
Lane
A
T
C
G
A
T
C
G
Removal
of T
Removal
of C
Removal
of G
Removal
of A
Electrophoresis and autoradiography
Cleavage at “base-less” site

116. Sanger used dideoxynucleo-
tides to sequence DNA
He had earlier devised a
protein sequencing method
Sanger’s dideoxy method
Frederick Sanger

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

118. Sanger’s method is based on controlled
interruption of replication
The DNA strand to be sequenced is used
as a template for replication
Replication is done in four test tubes

119. The following are added to each tube:
• The template strand
• 32P-Labeled primer
• dATP, dGTP, dCTP and dTTP
• DNA polymerase
• One dideoxynucleotide (ddATP,
ddGTP, ddCTP or ddTTP)

120. 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
Several cycles of replication are carried out

121. 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

122. After several cycles of replication,
strands of different lengths are formed
Each strand ends with a dideoxy-
nucleotide
Strands formed in the four tubes are
separated by electrophoresis
Electrophoresis is done in four parallel
lanes

123. The fragments are visualized by auto-
radiography
The pattern of bands on the autoradio-
gram gives the sequence of bases
This sequence is complementary to the
template strand

124. 8
7
6
5
4
3
2
1
3’
5’A
Lane
G
Lane
C
Lane
T
Lane
G
C
T
A
G
C
T
A
Electrophoresis and autoradiography
5’‒ATCGATCG
3’‒TAGCTAGC
Tube 1
5’‒ATCGATCG
3’‒TAGCTAGC
Tube 2
5’‒ATCGATCG
3’‒TAGCTAGC
Tube 3
5’‒ATCGATCG
3’‒TAGCTAGC
Tube 4
5’‒ATCGATCG
3’‒TAGCTAGC
5’‒ATCGATCG
3’‒TAGCTAGC
5’‒ATCGATCG
3’‒TAGCTAGC
5’‒ATCGATCG
3’‒TAGCTAGC
dGTP
dCTP
3’‒TAGCTAGC
5’■
dATP
dTTP
ddATP*
DNA
poly-
merase
3’‒TAGCTAGC
5’■A*
3’‒TAGCTAGC
5’■ATCGA*
+
3’‒TAGCTAGC
5’■ATCG*
+
3’‒TAGCTAGC
5’■ATCGATCG*
3’‒TAGCTAGC
5’■ATC*
+
3’‒TAGCTAGC
5’■ATCGATC*
3’‒TAGCTAGC
5’■AT*
+
3’‒TAGCTAGC
5’■ATCGAT*
dGTP
dCTP
3’‒TAGCTAGC
5’■
dATP
dTTP
ddGTP*
DNA
poly-
merase
dGTP
dCTP
3’‒TAGCTAGC
5’■
dATP
dTTP
ddCTP*
DNA
poly-
merase
dGTP
dCTP
3’‒TAGCTAGC
5’■
dATP
dTTP
ddTTP*
DNA
poly-
merase

125. Sanger’s method has also been auto-
mated
This has made DNA sequencing much
faster