RDT,Gene cloning, Restriction Endonucleases, Blunt end,Sticky end, DNA ligase, Vestors, PCR, DNA Polymerase, Bloting techniques, Application of Recombinant technology, Genomic Library, cDNA Library

1. Recombinant DNA
Technology
By
Vivek Chourasiya
M.Sc. Medical Biochemistry

2. What is Recombinant DNA
• Recombinant DNA technology, joining together of DNA
molecules from two different species that are inserted into
host organism to produce new genetic combinations.
• Thus the name recombinant DNA is also referred to as
“CHIMERIC DNA”.

3. History of Recombinant DNA
technology
• Recombinant DNA technology is one of the recent
advances in biotechnology, which was developed by two
scientists named Boyer and Cohen in 1973.

4. Gene cloning
• It can be defined as the isolation and amplification of an
individual gene sequence by insertion of that individual gene
sequence into a bacterium where it can be replicated

5. BASIC STEPS IN GENE CLONING
Step 1
• A fragment of DNA, containing the gene
to be cloned, is inserted into a circular
DNA molecule called a vector, to produce
a chimera or recombinant DNA (rDNA)
molecule.

6. Step 2
• The vector acts as a vehicle that
transports the gene into a host cell,
which is usually a bacterium although
other types of living cell can be used.
• This process is called transformation.

7. Step 3
• Within the host cell the vector multiplies producing numerous
identical copies not only of itself but also of the gene that it carries.

8. Step 4
• When the host cell divides, copies of
rDNA molecule are passed to the
progeny and further vector replication
takes place.

9. Step 5
• After large no: of cell divisions a colony or clone of identical host cells
is produced. Each cell in the clone contains one or more copies of the
rDNA molecule
Step 6
• Then, the host cells are then lysed and rDNA can be separated.

10. RESTRICTION ENDONUCLEASES
• These are the bacterial enzymes that can cut / split DNA at specific
sites.
• Found naturally in a wide variety of
prokaryotes
• An important tool for manipulating DNA.
• Ex: Eco R1, Hind 3.

11. HISTORY OF RESTRICTION ENDONUCLEASE
• First restriction enzyme was isolated in 1970 by
Hind ll.
• From then Over 3000 restriction enzymes have
been studied in detail, and more than 600 of these are
available commercially and are routinely used for DNA
modification and manipulation in laboratories.

12. NOMENCLATURE OF RESTRICTION ENZYME
• Each enzyme is named after the bacterium from which it was isolated
using a naming system based on bacterial genus, species and strain.
• For e.g EcoRI

13. Mechanism of Action
• Restriction Endonuclease scan the length of the DNA,
binds to the DNA molecule when it recognizes a specific
sequence and makes one cut in each of the sugar
phosphate backbones of the double helix – by
hydrolyzing the phoshphodiester bond.

14. •RECOGNITION SITE : It is the site where the DNA
is cut by a restriction endonuclease. Restriction
endonucleases can specifically recognize DNA
with a particular sequence of 4-8 nucleotides
and cleave.
•CLEAVAGE PATTERNS : The cut DNA fragments
may have sticky ends or blunt ends.

15. Ends Of Restriction Fragments
Blunt ends
Sticky ends

16. Blunt ends
• Some restriction enzymes cut DNA at opposite base
• They leave blunt ended DNA fragments
• These blunt ended fragments can be joined to any other DNA
fragment with blunt ends.

17. Sticky ends
• Most restriction enzymes make staggered Cuts
• Staggered cuts produce single stranded“sticky-ends”

18.  The base sequence recognised by a restriction
enzyme:
• Is 4-8 base pairs in length
• 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, MADAM, RADAR etc

19.  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
5’ GGCC 3’ 5’ TTTAAA 3’
3’ CCGG 5’ 3’ AAATTT 5’

20. • The cut DNA fragments are covalently joined together by DNA ligases.
• They form a phosphodiester bond between the phosphate group of 5’-
carbon of one deoxyribose with hydroxyl group of 3’- carbon of another
deoxyribose.
DNA LIGASES

21. Vectors
• Vector is an agent that can carry a DNA
fragment into a host cell in which it is
capable of replication.

22. Properties of a good vector:
• It should have ori region.
• It should contain at least one selectable marker
e. g. gene for antibiotic resistance.
• It should have unique restriction enzyme site
(only one site for one RE) for different REs to
insert foreign DNA.
• It should be preferably small in size for easy
handling.

23. Types of vectors
• Plasmid vectors
• Bacteriophage vectors
• Cosmids
• BACs & YACs

24. Plasmids
• Plasmids are extrachromosomal circular DNA
molecule that replicates inside the
bacterial cell.
• Cloning limit: 100 to 10,000 base pairs or 0.1-
10
kilobases (kb) .
• In their simplest form, plasmids contains a
bacterial origin of replication, an antibiotic
resistance gene, and at least one unique
restriction enzyme recognition site which helps
in
cloning.

25. Advantages of Plasmids in Molecular Biology
• Easy to work with - Plasmids are a convenient size
(generally 1,000-20,000 base pairs).
• Self-replicating - Endless number of copies of the
plasmid was obtained by growing the plasmid in
bacteria.
• Stable - Plasmids are stable long-term either as
purified DNA or within bacteria.

26. BACTERIOPHAGE
• Also known simply as a phage; a virus that
attacks and infects bacteria. The infection
may or may not lead to the death of the
bacterium, depending on the phage and
sometimes on conditions. Each
bacteriophage is specific to one form of
bacteria.
• Useful for cloning large
DNA fragments
(10 - 23 kbp)

27. INSERTION OF FOREIGN DNA INTO
BACTERIOPHAGE

29. LYTIC AND LYSOGENIC CYCLE

30. Cosmids
• Cosmids - an extrachromosomal circular DNA
molecule that combines features of plasmids and
phage; cloning limit - 35-50 kb
• Advantages:
▫ Useful for cloning very large DNA fragments
(32 - 47 kbp)

31. YAC & BAC
Advantages:
▫ Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
▫ This is very important for genome sequencing
projects
• Disadvantages:
▫ Not easy to handle extremely large DNA molecules

32. GENE AMPLIFICATION
Cell-based cloning: DNA is amplified in vivo by a cellular
host, such as a replicating bacteria (commonest),
baker’s yeast etc. The desired DNA template is introduced into a cellular
host. As the number of replicating host cells
increases (by cell divisions), the number of copies of the
desired DNA template also increases correspondingly.
Enzyme-based cloning: This method, represented by
polymerase chain reaction (PCR), involves in vitro
DNA amplification.

33. • This method requires separation of target DNA, its insertion into
vector to produce chimeric (recombinant)
• DNA, and introduction into host cell where it is amplified.
• It is a complex process which can be divided in the
• following major steps:
 Isolation of the target DNA.
 Introduction of target DNA into replicon.
 Transformation of the host cell.
 Isolation of DNA insert or its protein product.

36. What is PCR?
• PCR targets and amplifies
a specific region of a DNA
strand.
• It is an in-vitro technique
to generate large
quantities of a specified
DNA.
• PCR is ‘photocopier.’

37. HISTORY
• Kary B. Mullis,developed PCR in 1985
and was awarded the Nobel Prize for
Chemistry in 1993.
• PCR machine =thermocycler

38. Thermocycler

39. REQUIREMENT
• DNA Template
• Primers
• Taq polymerase
• Deoxynucleoside
triphosphates(dNTPs)
• Buffer solution
• Divalent
cations(eg.Mg2+)

40. STEP INVOLVED:

41. DENATURATION
• The reaction mixture is heated to a temperature between 90-98⁰ C so
that the ds DNA is denatured int single strands by disrupting the
hydrogen bonds between complementary bases.
• Duration of this step is 1-2 mins.

42. ANNEALING
• The annealing step allows the hybridisation of
the two oligonucleotide primers, which are
present in excess, to bind to their
complementary sites that flank the target
DNA.
• The annealed oligonucleotides act as primers
for DNA synthesis, since they provide a free 3’
hydroxyl group for DNA polymerase.
• TEMPERATURE :45-60°C

43. EXTENSION
• The temperature is now shifted to 72˚ C which is ideal for polymerase.
• Primers are extended by joining the bases complementary to DNA
strands.
• Elongation step continues where the polymerase adds dNTP’s from
5’to3’, reading the template from 3’to 5’ side bases are added
complementary to the template.
• Now first cycle is over and next cycle is continued , as PCR machine is
automated thermocycler the same cycle is repeated upto 30-40
times.

45. Taq DNA polymerase
• The availability of a thermostable DNA
polymerase enzyme isolated from the
thermophilic bacterium Thermus aquaticus
found in hot springs provided the means to
automate the reaction.
• Taq DNA polymerase has a temperature
optimum of 72°C and survives prolonged
exposure to temperatures as high as 96°C and
so is still active after each of the denaturation
steps.

46. Techniques for identification of DNA and RNA
• The technique for DNA identification is termed
Southern blot, whereas Northern blot is for RNA and
Western blot for protein identification.

47. Southern Blot Technique
• Invented by the English Molecular Biologist Edwin Southern in 1975.
• Determine the presence of a specific DNA sequence within a large,
complex DNA sample.
• Probe with radioactive DNA

48. STEPS OF SOUTHERN BLOTTING
• 1. Extraction of gene of interest from the human genome cell and
cutting it into fragments with the help of enzyme Restriction
endonuclease.
• 2.Electrophoresis – The separated fragments are then subjected to
agarose or polyacrylamide gel electrophoresis. The fragments are
separated by this method.
• 3.Denaturation- The double stranded DNA are melted to single
stranded by soaking it in strong alkali solution.

49. 4. Transfer – Single stranded DNA are transferred to the sheet of nitro
cellulose filter.
This is referred to as blotting.
This nitrocellulose immobilizes the single stranded DNA.
5.Addition of nucleotide Probe –It allows hybridization of probe with
the matching DNA fragment.
Generally radioactive probes are used.
6.Autoradiograhy : Determines the position of the labelled probe with
the sequence of interest.

50. SOUTHERN BLOT TECHNIQUE

51. Northern Blot Technique
• This technique is developed by James Alwine, David Kemp, and
George Stark in 1977.
• Which is similar to the Southern blot technique.
• Detect the presence a specific mRNA in a total RNA extract.

52. Western Blot Technique
• Developed by W. Neal Burnette in 1981
• It is also known as immunoblot.
• Detects the presence of specific protein
• Protein probed with radioactive or enzymatically – tagged antibodies.

53. Two similar methods used for determining the
order of the nucleotide sequences:
• To determine the order of the nucleotide bases adenine, guanine,
cytosine, and thymine in a molecule of DNA two methods were used
1. Maxam and Gilbert; Chemical Sequencing
2. Sanger; Chain Termination Sequencing

54. Applications of rDNA technology
• Manufacture of proteins/hormones , plasminogen activating factor,
blood clotting factors, insulin, growth hormone.
• AIDS test: Has become simple & rapid
• Diagnosis of molecular diseases: sickle cell anaemia, thalassaemia,
familial hypercholesterolaemia, cystic fibrosis
• Prenatal diagnosis: DNA from cells collected from amniotic fluid

55. • Gene Therapy:
This is achieved by cloning a gene into a vector that will readily be
taken up & incorporated into genome of a host cell. ADA deficiency has
been successfully treated Application in
• Agriculture:
Genetically engineered plants are developed to resist draught &
diseases. Good quality of food & increased yield of crops is also
possible.

56. • Industrial Application:
Enzymes---use to produce sugars, cheese, detergents. Protein
products---used as food additives, increases nutritive value, besides
imparting flavour.
• Application in forensic medicine:
The restriction analysis pattern of DNA of one individual will be very
specific(DNA fingerprinting),but the pattern will be different from
person to person. Helps to identify criminals & to settle disputes of
parenthood of children.

57. DNA libraries –a collections of DNA
sequences.
 DNA libraries, like conventional libraries, are used to collect and
store information.
 In DNA libraries, the informationis stored as a set of DNA
molecules.
 All DNA libraries are collections of DNA fragments that represent a
particular biological system of interest.
 The two most common uses for these DNA collections are DNA
sequencing and gene cloning.

58. Types of DNALibraries
• The genomic library contains DNA representing the
entire genome of an organism.
• The cDNA library contains only complementary
DNA molecules synthesized from mRNA molecules in a cell.

59. Genomic Library
 Are made from total nuclear DNA of an organismor species.
 DNA is cut into clonable size pieces as randomly as possible
using restriction endonuclease.
 Genomic libraries contain whole genomic fragments including gene
exons and introns, gene promoters, intragenic DNA, centromeric
DNA, origins of replication, etc

60. Constructing libraries of clones
 The library is made by inserting these millions of fragments of
DNA into λ bacteriophage plasmids.
 This allows the genes to be grown up (cloned) in E. coli.
 The library can be screened for DNA fragments or Particular
genes

62. cDNALibrary
• The advantage of cDNA library is that it contains only the coding
region of a genome.

64. Vectors for DNALibraries
• Genomic libraries
 – λ-phage - 9-23 kb → convenient and easy to handle
 – Cosmids - 30-45
 – PAC, BAC, YAC →artificial chromosomes, accommodate
large fragments
• cDNAlibraries
 -λ-phage - 9-23 kb → provides selection for longer cDNAs
 -conventional plasmids → high level of expression of proteins.

65. ThankYou