it is the fundamentals of biotechnology coarse lecture slides

1. 4.Recombinant DNA Technology
• Tools of Recombinant DNA Technology
• Making Recombinant DNA
• DNA Library
• Transgenic
• Identification of Recombinants
• Polymerase Chain Reaction (PCR)
• DNA Probes
• Hybridization Techniques
1

2.  Recombinant DNA technology
• A technology which allows DNA to be produced via artificial means.
• It only becomes useful when that artificially-created DNA is reproduced (cloned).
• Genetic recombination is the exchange of information between two DNA segments.
• This is a common occurrence within the same species.
• But by artificial means, when a gene of one species is transferred to another living organism, it is called
recombinant DNA technology (rDNA).
• In common, this is known as genetic engineering.
eg. Combining DNA from human and bacteria
2
 Recombinant DNA Technology

3. • Recombinant DNA technology is one of the recent advances in biotechnology,
which was developed by two scientists named Boyer and Cohen in 1973.
• Recombinant DNA, having unrelated genes, is also known as chimeric DNA
• Recombinant DNA technology has proved to be of immense value in:
• Medical science
• Agriculture
• Animal husbandry
• Industry
3

4. 4

5. Tools of recombinant DNA technology
The basic tools for making rDNA molecules are:-
 Restriction endonucleases (Restriction Enzymes) or (molecular scissors) -
DNA cutting Enzymes
 Vectors- cloning vehicle - carry rDNA into a host cell for multiplication.
 Host cell- the factories of cloning
 DNA -Ligase – the connector of DNA fragments
5

6. 6
Restriction endonuclease
•Is an enzyme that cuts double-stranded or single stranded DNA at specific
recognition nucleotide sequences known as restriction sites.
•These are the bacterial enzymes that can cut/split DNA (from any source) at specific
sites and used as defense mechanism against invading viruses.
•They were first discovered in E.coli restricting the replication of bacteriophages, by
cutting the viral DNA (The host E. coli DNA is protected from cleavage by addition of
methyl groups).
•Thus, the enzymes that restrict the viral replication are known as restriction enzymes
or restriction endonucleases.

7. 7
Recognition sequence
 Is the site where DNA is cut by a restriction endonuclease
 Restriction endonucleases can specifically recognize DNA with a particular
sequence of 4-8 nucleotides & cleave
 Cleavage patterns:- Majority of restriction endonucleases (particularly type II) cut
DNA at defined sites within recognition sequence.
 DNA fragments with sticky ends are useful for recombinant DNA experiments.
 This is because the single-stranded sticky DNA ends can easily pair with any
other DNA fragment having complementary sticky ends.

8. 8
Recognition sequences are palindromic
 A palindrome is a word or a sentence which reads the same from left to right and
from right to left example:- DAD, MADAM, RADAR or the base sequences read
the same backwards and forwards.
 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
 Example
5’ GGCC 3’ 5’ TTTAAA 3’
3’ CCGG 5’ 3’AAATTT 5’

9. 9
In theory, there are two types of palindromic sequences that can be possible in DNA
The mirror-like palindrome in which a sequence reads the same forward and
backwards on the same DNA strand (i.e., single stranded) as in GTAATG.
The inverted repeat palindrome is also a sequence that reads the same forward
and backwards
But the forward and backward sequences are found in complementary DNA
strands (i.e., double stranded) as in GTATAC (Notice that GTATAC is
complementary to CATATG).
 The inverted repeat is more common and has greater biological importance than the
mirror-like.

10. 10
Nomenclature of restriction enzyme
Smith and Nathans (1973) proposed enzyme naming scheme
Based on the origin of the bacteria they extracted from
First letter: initial letter of the genus name of the organism from which the
enzyme is isolated
Second and third letter: usually initial letters of the organism’s species name
Fourth letter (if any): indicates a particular strain of organism
Roman numerals: indicate the order of discovery of enzymes from the same
organism and strain

11. 11
EcoRI
 E = genus Escherichia
 co = species coli
 R = strain RY13
 I= first endonuclease isolated
BamHI
 B = genus Bacilus
 am = species amyloliquefaciens
 H = strain H
 I = first endonuclease isolated
HindIII
H = genus Haemophilus
in = species influenzae
d = strain Rd
III = third endonuclease isolated

12. 12
 Types of RE
Type I
Are less specific (cut DNA randomly).
Their recognition and cleavage sites are different.
Cleave DNA at about 1000bp away from recognition site.
Eg. EcoK. have recognition sequences of about 15b.
Type II
Is the most commonly used RE.
Have cleavage site within the recognition sequences.
The recognition sequences form palindromes (read the same from left to right and vice
versa).
E.g EcoRI NNNNNNNG’AATTCNNNNNNNN
NNNNNNNCTTAA’GNNNNNNNN
Most type II REs have recognition sequences of 4- 8 bp.

13. 13
Type III
 Cleave DNA in immediate vicinity of their recognition sites
 These are also less specific
E.g EcoPI
Type I and Type III REs are not used for gene cloning since they cannot cut DNA
precisely. i.e. they cleave DNA randomly
Type IV
Cleave only modified DNA (methylated, hydroxyl methylated and glucosyl-hydroxyl
methylated bases).
Recognition sequences have not been well defined
Cleavage takes place ~30 bp away from one of the sites.
Sequence similarity suggests many such systems in other bacteria and archaea.

14. 14
Examples of Type II RE used in rDNA experiments

15. 15

16. 16
Host cells – the factories of cloning
 The hosts are the living systems or cells in which the carrier of recombinant
DNA molecule or vector can be propagated.
 There are different types of host cells prokaryotic (bacteria) & eukaryotic (fungi,
animals & plants).
 Microorganisms are preferred as host cells, since they multiply faster compared
to cells of higher organisms (plants or animals).

17. 17
Prokaryotic hosts
 Escherichia coli:-
 Escherichia coli was the first organism used in the DNA technology & continues to be the host of
choice by many workers.
 The major drawback is that E. coli (or even other prokaryotic organisms) cannot perform post-
translational modifications.
 Bacillus subtilis as an alternative to E.coli.
 Eukaryotic hosts
 The most commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae.

18. 18
 Certain complex proteins which cannot be synthesized by bacteria can be produced
by mammalian cells e.g. tissue plasminogen activator (clot dissolving protein).
 The mammalian cells possess the machinery to modify the protein to the active
form (posttranslational modifications).
 DNA Ligase
 The cut DNA fragments are covalently joined together by DNA ligases.
 These enzymes were originally isolated from viruses.
 They also occur in E.coli & eukaryotic cells.
 DNA ligases actively participate in cellular DNA repair process.

19. 19
DNA vectors -Cloning Vehicles
 To transfer genes, there should be a mechanism of transport
Vectors are DNA molecules that serve as vehicles to carry gene in to cell.
It is capable of replicating in a suitable host cell.
The foreign DNA is also multiplied.
 Cloning vectors:- Vectors used only for the multiplication of the gene of interest
 Expression vectors:- Vectors used for expressing the gene of interest in the host cell
 It is also possible that a vector can simultaneously serve as a cloning and expression
vector.

20. 20
Features of A Cloning Vector
 All commonly used cloning vectors have some essential
features:
 Origin of replication (ori):
 This makes autonomous replication in vector.
 Ori is a specific sequence of nucleotide from where
replication starts.
 When foreign DNA is linked to the sequence along with
vector replication, foreign (desirable) DNA also starts
replicating within host cell.
Multiple Cloning Site( polilinker):
 Cloning site is a place where the vector DNA can be
digested
 and desired DNA can be inserted by the same restriction
enzyme.
 It is a point of entry or analysis for genetic engineering
work.
 Recently recombinant plasmids contain a multiple
cloning site (MCS) which have many (up to ~20)
restriction sites.

21. 21
 Selectable Marker
 Selectable marker is a gene that confers
resistance to particular antibiotics or selective
agent that would normally kill the host cell or
prevent its growth.
 A cloning vector contains a selectable marker,
which confer on the host cell an ability to survive
and proliferate in a selective growth medium
containing the particular antibiotics.
 Reporter Gene or Marker Gene
 Reporter genes are used in cloning vectors to
facilitate the screening of successful clones by
using features of these genes that allow successful
clone to be easily identified.
 Such feature present in cloning vectors is used in
blue-white selection.

22.  Cloning vectors can be plasmids, bacteriophage, viruses, or even small artificial chromosomes.
 Most vectors contain sequences that allow them to be replicated autonomously within a compatible
host cell, whereas a minority carry sequences that facilitate integration into the host genome.
 All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a
restriction endonuclease to allow site-specific insertion of foreign DNA.
 The most useful vectors have several restriction sites grouped together in a multiple cloning site
(MCS) called a polylinkers.
22

23. Requirements of a vector to serve as a carrier molecule
 The choice of a vector depends on the design of the experimental system and how the cloned
gene will be screened or utilized subsequently
 Most vectors contain a prokaryotic origin
 Some vectors contain an additional eukaryotic origin
 Multiple unique cloning sites are important for versatility and easier library construction.
 Antibiotic resistance genes and/or other selectable markers enable identification of cells that
have acquired the vector construct.
 Must be small in size and non-pathogenic
23

24. Type of vectors
The most commonly used vectors in recombinant DNA are:
 Plasmids
 Bacteriophages
 Cosmids
 Shuttle vectors
 Artificial chromosome vectors
 bacterial artificial chromosome (BACs)
 yeast artificial chromosome (YACs)
 Human artificial chromosomes (HACs)
24

25. Plasmids
• Are extrachromosomal, double stranded, circular, self-replicating DNA molecules.
• Plasmids are found in almost all bacteria
• These extra chromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells
exist in a parasitic or other symbiotic relationship with their host cell.
• Can replicate autonomously within a host
• They frequently carry genes that cause resistance to antibiotics such as tetracycline, ampicillin,
or kanamycin.
• The expression of these marker genes can be used to distinguish between host cells that carry
the vectors and those that do not.
25

26.  Plasmids are not essential for the survival of bacteria
 They replicate independently from the bacterial chromosome
 They carry information that may give the respective bacteria an advantage over other bacteria
 Antibiotic resistance, metabolic activities and virulence factors
 The ability to join with other bacteria for the exchange of genetic material
 Their size ranges from 1 – 200 kb
 Plasmids to be used as DNA vectors should have marker genes for the identification of
 Successful insertion of the foreign DNA
 Successful transfer to the recipient cell
26

27. Are present in low (1- 4) or high (10- 100) copy number per cell
Confer drug resistance to bacterial strains.
Contain their own origin of replication (ORI).
Can only carry foreign DNA less than 8 kb
Advantages:
Small, easy to handle
Straightforward selection strategies
Useful for cloning small DNA fragments (< 10kbp)
Disadvantages:
Less useful for cloning large DNA fragments (> 10kbp)
27

28. Nomenclature of Plasmids
 No internationally standardized rules, but certain habits are generally followed
 p plasmid
 ABC 2-3 letters, usually the initials of the plasmid‘s creator
 123 2-3 figures, indicating the serial number of this particular plasmid in the lab‘s collection
pBR322 = plasmid 322 from the collection of Paco Bolivar and Ray Rodriguez
pUC18 = plasmid 18 from the University of California collection
28

29. 29
 The name ‘pBR322’ conforms with the standard
rules for vector nomenclature.
 ‘p’ indicates a plasmid.
 ‘BR’ identified the laboratory in which the vector
was originally constructed (BR stands for Bolivar
and Rodriguez the two researchers who
developed pBR322)
 ‘322’ distinguishes this plasmid from others
developed in the same laboratory (there are also
plasmid called pBR325, pBR327 etc.)
 The Nomenclature of Plasmid Cloning Vector

30. pBR322
 One of the original plasmids based on ColE1 plasmid
 Size is relatively small: 4,361 bp
 About 6 kb of inserts can be added
 Contains the ori from ColE1
 The normal copy number of 15-20 per cell can be increased to 1000-3000 through
chloramphenicol application
 Carries two antibiotic resistance genes with many restriction sites for easy identification
of clones 30

31. 31

32. pUC19
 Derivative of pBR322
 were a major step forward as they allowed screening in a single step
 the ampicillin resistance gene from pBR322 remains, but was modified by removing many of
the restriction sites
 Have major new features
 Smaller – so can accommodate larger DNA fragments during cloning (5-10kbp)
 instead of tetracycline resistance it contains the lacZ gene (β-galactosidase)
 a mutation in the ori allows for numbers of 500-600 per cell without the supplementation of
chloramphenicol
 Multiple cloning sites clustered in same location “polylinker” into the lacZ gen
32

33. Bacteriophage (Lambda vector)
• Bacteriophage lambda (λ) infects E. coli
• Double-stranded, linear DNA vector – suitable for library construction
• use only 50 % of the DNA for replication, the rest can serve as cloning sites.
• Can accommodate large segments of foreign DNA
• Central (1/3 size) = “stuffer” fragment
• Can be substituted with any DNA fragment of similar size without affecting ability
of lambda to package itself and infect E. coli
• Accommodates ~15kbp of foreign DNA
33

34.  Foreign DNA is ligated to Left and Right Arms of lambda
 Transfected into E. coli as naked DNA, or
 Packaged in vitro by combining with phage protein components (heads and tails) (more efficient,
but labor intensive and expensive)
 of all phages, only λ (lambda) and M13 are widely used for biotechnology applications
34

35. Cosmid vectors
 Hybrid molecules containing components of both lambda and plasmid DNA
Lambda components: COS sequences (required for in vitro packaging into phage
coats)
Plasmid DNA components: ORI + Antibiotic resistance gene
 possess the characteristics of both.
 can be packed as phages and inserted in to E. coli.
 After infection of E. coli, rDNA molecules replicate as plasmids
35

36. Very large inserts can be accommodated by cosmids (up to 35-45 kbp)
Disadvantages:
Not easy to handle very large plasmids (~ 50 kbp)
36

37. Shuttle vectors
 Hybrid molecules designed for use in multiple cell types
 Multiple ORIs allow replication in both prokaryotic and eukaryotic host cells allowing
transfer between different cell types
E. coli  yeast cells
E. coli  human cell lines
Bacterial artificial chromosomes (BACs)
 Based on F factor of bacteria
 Can accommodate 1 Mb of DNA (= 1000kbp)
 F factor components for replication and copy # control are present
 Selectable markers and cloning sites available
37

38. BAC vector
38
 oriS and RepE mediate replication
 parA and parB maintain single copy
number
 Chloramphenicol Resistant marker

39. Yeast artificial chromosomes (YACs)
 Hybrid molecule containing components of yeast, protozoa and bacterial plasmids
Yeast:
ORI = ARS (autonomously replicating sequence)
Selectable markers on each arm (HIS3 and URA3)
Yeast centromere
Protozoa= Tetrahymena
Telomere sequences Bacterial plasmid
Polylinker
 Can accommodate >1Mbp (1000kbp = 106 bp)
39
telomere telomerecentromere
URA3ARS HIS
3
replication
origin
markers
large
inserts

40. Expression vectors
Expression of foreign DNA in a host cell from different origins is difficult.
 because the host cell expression machinery does not recognize the expression signals of the
foreign DNA.
 put the foreign DNA under the control of the host cell’s expression signals.
 Thus it is necessary to insert regulatory elements (signals) in to the vector along with the gene of
interest to make it an expression vector.
 Eg. rDNA of human insulin gene, regulated by bacterial host cell
 Expression vectors must have
 promoter and terminator sequences: for initiation and termination of transcription.
 start codon and a ribosome binding site: for translation initiation
40

41. Making the recombinant DNA
 In order to make the recombinant DNA, 3 steps are required
 Extraction of DNA, checking for purity and quantification
 Generating DNA fragments
 Insertion of DNA fragments in to the vector
Extraction of DNA, quantification and checking for purity
Basic DNA extraction technique
 The use of DNA for analysis or manipulation usually requires the isolated DNA to be purified
to a certain extent.
 Depending on the type of cells or tissues there are a number of standardized techniques
41

42. 42
In any method of extraction and purification, there are 3 main steps
Rupturing/ lysis of cells
 Mechanical Method by Rapid freeze drying of tissues in liquid Nitrogen (-196 0C)
 Chemical Method Detergents such as Sodium dodecyl sulphate
 Enzymatic method Employs lysozyme
Purification of DNA
 Pronase or Proteinase K enzymes that hydrolyses polypeptides to smaller units
 Phenol Extraction using solvents with different constituent
Quantitation and checking for purity
 UV spectrophotometer
 Gel electrophoresis

43. Generating DNA fragments
 The gene of interest is found in the extracted DNA.
 Ex. The human insulin gene is found in the human genome
 cleave the DNA in to many small fragments.
 b/c the whole genome is too large to insert in to the vector
 Ex. Plasmids can only clone DNA less than 8 kb
 The DNA can be precisely fragmented using RE
 Thus from among the fragments, one will carry the gene of interest.
43

44.  It is not just the genome that should be fragmented, but also the vector that carries the gene of
interest.
 The same RE should be used to fragment the genome and the vector.
 Since a RE recognizes a certain restriction site and cuts DNA at only a specific cleavage site, similar
cut ends that are complementary can be produced both on the vector and the genome fragments.
44

45. 45

46. Insertion of DNA fragments in to the vector
 DNA from the genome and vector may be complementary and are thus attracted to each other by H
bond.
 stable rDNA molecule can be produced by forming phosphodiester bond between the fragments
 The vector and the gene of interest are ligated together by DNA ligase.
 The enzyme can be isolated from virus, bacteria (E. coli) and eukaryotic cells.
46

47. 47
Making rDNA and cloning

48. Isolate plasmid DNA
and human DNA.
Cut both DNA samples with
the same restriction enzyme.
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many non recombinant plasmids.
Bacterial cell lacZ gene
(lactose
breakdown)
Human
cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid Gene of
interest
Sticky
ends
Human DNA
fragments
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria
48

49. Constructing Genomic and cDNA library
 Entire genome fragmented using REs and the fragments make different rDNAs.
 A collection of rDNAs representing all the genes/ sequences in the entire genome
 Then each rDNA carrying a fragment is inserted in to a host cell and as the cells divide,
the rDNA is cloned.
 A collection of cells is created composed of fragments that represent all sequences including
all the genes in the entire genome.
 This collection is called genomic library.
49

50.  From among the clones, any gene can be withdrawn from the library for further study.
 Therefore, the gene of interest can be isolated and identified from the genomic library.
 For example, the human genomic library contains all the genes of the human genome
distributed as fragments in different clones.
 Among the clones, one will carry insulin coding gene which can be identified by
screening methods.
50

51. 51

52. λ replacement vector cloning
3. Packing with a mixture of the
phage coat proteins and phage DNA-
processing enzymes
4. Infection and formation of
plaques
Library constructed
2. Ligation
1. preparation of arm and genomic
inserts
52

53.  Eukaryotic genes usually contain non-coding regions, introns.
 The genes with introns can be expressed in prokaryotic systems as they lack the splicing
machinery.
 So the gene will be expressed along with the introns and the result will be an entirely different
protein, which is not active or functional.
53

54.  Therefor instead of entire DNA sequence, possible to use cDNA derived from mRNA
 cDNA library is a collection of clones of DNA, which are the complementary copies mRNA isolated
from the respective cells.
 Processed mRNA is the starting material for the construction of the cDNA.
 Since the mRNAs are produced after splicing, they are devoid of introns.
 it is necessary to identify and extract mRNA from a cell or a tissue
 reverse transcriptase uses mRNA as a template to synthesize the DNA
 cDNA represents only exons, coding regions of the actual eukaryotic genes.
 This cDNA can be stored in plasmids or phages
54

55. INTRODUCTION OF RECOMBINANT DNA INTO HOST CELLS
 After the construction of the recombinant DNA molecule, it has to be introduced into a
suitable host cell so that the DNA will multiply
 Several methods available depending on several factors including the type of vector used
and the host cell
 The most common methods used for introducing the rDNA molecule into the host cell
are:
Transformation
 The most commonly adopted method for introducing a recombinant DNA into a host cell
 Some cells take up foreign DNA from their environment But many cells including E.
coli, yeast, and mammalian cells are not able to take up the DNA from their
environment naturally.
55

56.  Certain chemical treatments can enhance the ability of cells to take up the foreign DNA or make
the cell competent for transformation
 This enhanced competency for transformation was found out by Mandel and Higa in 1970
 They observed that e. coli cells become more competent to take up foreign DNA when these cells
are incubated in cold calcium chloride solution
 This mechanism is still followed for the transformation of e. coli cells as a part of gene cloning.
56

57.  Transfection
 Method used for the transformation of cultured cells
 Recombinant vector is mixed with charged substances such as calcium phosphate, or cationic
liposomes and Over layered on the host cells
 This ultimately results in the uptake of the external DNA by these host cells
 Electroporation
 Efficient method for introducing rDNA into a host cell
 Electric current is used to create temporary microscopic pores in the cell membrane of the host cell
 Through these temporary openings foreign DNA enters the cell
 Especially suitable for cells such as yeast, mammalian cells, and plant protoplasts.
57

58. Microinjection
 Specialized technique by which the DNA fragment or gene can be directly injected into the
nucleus of plant and animal cells
 can be carried out without the use of any specialized vector
 involves the direct injection of the DNA into the nucleus of the host cell with the help of a glass
microinjection tube or syringe
The biolistic method
 was developed for introducing foreign DNA into plant cells with the help of a gene or particle
gun
 Microscopic particles of gold or tungsten coated with the DNA of interest is bombarded into the
cells at a high velocity
 The bombardment of the particle is carried out with the help of a mechanical device called a
particle gun
58

59. IDENTIFICATION OF RECOMBINANTS
 In the process of making the rDNA for constructing the genomic/ cDNA libraries, ligation may not always be successful.
 In some cases,
 the vectors and DNA fragments remain unligated
 or else, the vector may simply self ligate.
 During transformation some host cells may not take up the rDNA (untransformed cells).
59

60.  There are various selection/screening techniques
 Based on the expression or non-expression of some traits.
 Genetic markers are commonly used for screening
 Two major methods of selection of recombinant cell using these markers are:
 Selection of plasmid vectors carrying antibiotic resistance genes.
 Lac selection/Blue white selection/inactivation of beta-galactosidase activity.
60

61. Screening technique through drug resistance
 pBR322 contains two antibiotic resistant genes
 Ampicillin resistance gene (ampR) that codes for the enzyme β-lactamase, which detoxify
ampicillin
 Tetracycline resistance gene (tetR) coding for sets of enzymes that detoxify tetracycline.
 Both these genes can serve as cloning sites.
 However for the purpose of selection during insertion of the gene of interest in to the vector, only
one of these genes is selected as the cloning site.
o Eg, if ampR gene is chosen as a cloning site, the cell is susceptible to ampicillin and resistant
to tetracycline
 But inappropriate recombinant, untransformed cells and self ligated vector will be resistant to both
61

62. 62

63. Procedure of Screening
 The vector, pBR322, containing ampR and tetR genes are used for cloning.
 Only the ampR gene is used as a cloning site and cut with the RE to insert the DNA fragments.
 Presumably transformed cells are cultured on two media:
 Medium I containing Tetracycline (control medium)
 Medium II containing tetracycline and ampicillin
 The cells are first cultured on medium I. Then a replica plate is made on medium II directly
from medium I.
63

64. Analysis of results
The resulting colonies of cells on both media are analyzed as follows:
 Cells not been transformed (contain no pBR322 at all) will be susceptible to amp & tet and thus
will not survive in both media.
 Transformed Cells with non recombinant pBR322 that has been self ligated will be resistant to
both amp and tet. Therefore, these cells will survive in both media I and II.
 Cells that contain the recombinant plasmid will be resistant to tetracycline and grow on Medium I
but are susceptible to amp and therefore will not survive on Medium II.
 These cells will be selected from the control medium (medium I) as the recombinant cells.
64

65. 65

66. Screening for recombinant cells carrying the gene of interest
 Within the genomic or cDNA library, cells carrying the gene of interest can be selected by
 polymerase chain reaction (PCR) technique,
Polymerase Chain Reaction (PCR)
 It is a technique of synthesising multiple identical copies of a gene by replication of DNA in-
vitro.
 Developed by Kary Mullis in 1984 and has been a basic tool in a molecular biology laboratory.
 The cellular machineries and molecules for DNA replication are used for PCR amplification
66

67. Ingredients of a PCR mix
1. dsDNA as templates.
2. Two primers (forward and reverse),
3. Four deoxyribonucleotides
• dATP, dCTP, dGTP, dTTP
4. A thermostable DNA polymerase
• Stable at high temperature
• eg. Taq DNA polymerase isolated from Thermus aquaticus (a bacterium found in hot
springs)
67

68. Stages in PCR
Denaturation
 Separation of the dsDNA by heating (94 0C) for 1 minute.
 The temperature breaks the H bonds and each strand (ssDNA) acts as a template.
Annealing
 Conducted at lower temperature (50- 60 0C) for 1 minute
 Allows the primers to attach to the flanking regions (DNA adjacent to 5’ end) of the gene of interest
Extension
 Conducted at 72-75 0C for 2 minutes, an optimal temperature for Taq polymerase
 DNA polymerases recognise the primers at the start tags and assemble complementary nucleotides.
68

69. 69

70.  If the gene of interest is not present in the cell, the primers cannot anneal with the DNA and
thus there will be no amplification of DNA.
 However, if present, the specific primers anneal and allow the amplification of the gene of
interest.
 When the products of PCR reactions are run on the gel, if the gene of interest is present and
thus amplified, a band is observed at a position expected to be the size of the gene.
 If the gene of interest is not present, no band is observed at the expected position.
70

71. 5. Biotechnology in Health
• Hybridoma Technology and Monoclonal Antibodies
• Application of Biotechnology in Medicine
Diagnosis
Vaccine production
Gene Therapy
Organ/tissue transplantation
Forensic medicine
Stem cell research and its potential in medicine
71

72. Hybridoma Technology and Monoclonal Antibodies
 Monoclonal Antibody Production technology was developed in 1975.
 Produced by cell lines or clones obtained from the immunized animals with the substance to be
studied
 Cell lines are produced by fusing B cells (spleen cell) from the immunized animal with myeloma
cells
 The fused product of a hybrid cell called a hybridoma and the technology that produce those
fused hybrid cells called hybridoma technology
 To produce the desired mAb, the cells must be grown in either of two ways
 injection into the peritoneal cavity of a suitably prepared mouse
 in vitro tissue culture. 72

73. Monoclonal Antibody
 Antibody from a single antibody producing B cell and therefore only binds with one unique epitope (part
of antigen where antibody attach itself).
 B-cell is isolated and fused to an immortal hybridoma cell line so that large quantities of identical antibody
can be generated.
 Advantages
 Can produce large quantities of identical antibody and have Batch to batch homogeneity.
 High specificity to a single epitope and Reduced probability of cross reactivity.
 Disadvantages
 Significantly more expensive to produce
 Requires significantly more time to produce and develop the hybridized clone
 Cell culture and purification capabilities required 73

74. Polyclonal Antibody
 collection of antibodies from different B cells that recognize multiple epitopes on the same
antigen
 Each of these individual antibodies recognizes a unique epitope that is located on that antigen.
 Advantages
 Inexpensive and quick to produce (Purified antibody ready to use in under four months)
 Higher overall antibody affinity against the antigen due to recognition of multiple epitopes.
 Offers greater sensitivity for detecting proteins that are present in low quantities in a sample
since multiple antibodies will bind to multiple epitopes on the protein.
 Disadvantages
 Variability between different batches produced in different animals at different times
 Higher potential for cross reactivity due to recognizing multiple epitopes
74

75. 1975, by Georges Köhler and Cesar Milstein
- Be awarded a Nobel Prize in1984
75

76. Formation and Selection of Hybrid Cells
 Hybridoma: the B cell X myeloma cell
 To be produce by using polyethylene glycol (PEG) to fuse cells
 The myeloma cells: immortal growth properties
 The B cells: to contribute the genetic information for synthesis of specific antibody
 Selected by using HAT medium (hypoxanthine, amino protein, and thymidine)
 Myeloma cells are unable to grow
 B cells are able to survive, but can not live for extended periods
76

77. Low concentration
(1-20 μg/ml)
High concentration
(1-10 mg/ml)
77

78. Application of Biotechnology in Medicine
Diagnosis
 Accurate diagnosis of disease is critical for effective management and cure.
 The standard procedure for detecting some pathogens is to grow it in the lab from clinical
specimens
 many viruses and some bacteria are difficult or even impossible to grow in the laboratory
 different microorganisms require different culture media and culture conditions
 Thus traditional methods of identification can be laborious.
 In contrast, molecular approaches analyze molecules such as DNA, RNA, or protein rather than
attempting to grow the disease casing agents.
 In some cases, it is quicker and more accurate than conventional methods.
78

79. Molecular diagnosis using DNA
 Every species of organism has a unique small-subunit ribosomal RNA coding sequence/ gene
(16S rRNA coding gene in bacteria, 18S rRNA coding gene in eukaryotes).
 Hence bacteria and eukaryotic parasites may be identified by analysis of this sequence.
 The method of analysis can perform by using PCR
79

80. Vaccine production
Recombinant vaccines
 developed from an antigen extracted and isolated from a pathogenic organism to cause an
immune response.
 Using recombinant DNA technology, it is possible to engineer organisms to produce the
antigen.
 DNA vaccines
 Instead of using an antigen for vaccination, a vector DNA (usually a plasmid) carrying a an
antigen coding gene can be administered in to the cells of the individual to be vaccinated.
 In the vaccinated individual, the gene is expressed to produce the immunogenic protein that
causes an immune response.
80

81. Approaches for introducing DNA vaccines
 Direct gene transfer in vivo
 It involves the injection of the plasmid vector into muscle
 Without any special delivery system, the plasmid is taken up and expressed in the muscle
cells.
 Particle gun delivery of recombinant plasmid DNA
 plasmid is coated with gold or tungsten particles and introduced into skin cells by a particle
gun
81

82. 82

83.  Gene Therapy
 Human beings suffer from more than 5000 different genetic diseases.
 Eg. Cystic fibrosis (inherited disorder affect work of organs like lung), sickle cell anemia
(inherited red blood cell disorder), hemophilia (blood clotting disorder), etc.
 In addition many common disorders like cancer, hypertension, mental illness… etc have genetic
components.
 Gene therapy is the introduction of a normal functional gene into cells, which contains the
defective allele of concerned gene with the objective of correcting a genetic disorder.
Types of gene therapy
 Germline gene therapy
 Somatic cell gene therapy
83

84. Germline gene therapy
 Germ cells i.e. sperms and eggs are modified by introduction of functional genes, which are
ordinarily integrated into their genomes.
 A fertilized egg is provided with a copy of the correct version of the relevant gene and re-
implanted into the mother.
 If successful, the gene is present and expressed in all cells of the resulting individual.
 Therefore the change due to therapy would be heritable and would be passed on to later
generations.
 not yet applied for human beings due to lack of development of the techniques and ethical issues.
 Used for curing diseases in animals (lower high blood pressure in rats)
84

85. Somatic gene therapy
 Somatic cell therapy involves manipulation of somatic cells
 Expression of the introduced gene eliminates symptoms of the disorder and is not heritable.
 Somatic cell therapy has potential in the treatment of
 Hemophilia
 Cystic fibrosis
 Pancreas failure
85

86. Mode of delivery of the functional gene
o Virus mediated
 Employs recombinant retroviruses or adenoviruses that carry the functional
gene and express the functional or correct gene in the host cell
o Non viral methods
 Naked DNA insertion
 Liposomes
 Electroporation
86

87. Organ/tissue transplantation
 organ transplant an operation moving an organ from one organism (the donor) to another (the
recipient)
Types of transplants
 Autograft
 A transplant of tissue from one to oneself
 Sometimes this is done with surplus tissue, or tissue that can regenerate, or tissues more
desperately needed elsewhere
 Eg. skin grafts
 Allograft
 Is a transplanted organ or tissue from a genetically non-identical member of the same
species
 Most human tissue and organ transplants are allografts. 87

88.  Isograft
 A subset of allografts in which organs or tissues are transplanted from a donor to a genetically identical
recipient (such as an identical twin).
 Xenograft
 A transplant of organs or tissue from one species to another.
 often an extremely dangerous type of transplant.
• Eg porcine heart valves, (successful), a baboon-to-human heart (failed), and piscine-primate (fish
to non-human primate)
 Split transplants
 Sometimes, a deceased-donor organ (specifically the liver) may be divided between two recipients,
especially an adult and a child.
88

89. Domino transplants
This operation is usually performed for cystic fibrosis as both lungs need
to be replaced and it is a technically easier operation to replace the heart
and lungs en bloc (all the same time).
As the recipient's native heart is usually healthy, this can then itself be
transplanted into someone needing a heart transplant
89

90. Major organs and tissues transplanted
Thoracic organs
 Heart (Deceased-donor only)
 Lung(Deceased-donor and Living-Donor)
 En bloc Heart/Lung (Deceased-donor and
Domino transplant)
 Other organs
 Kidney (Deceased-donor and Living-Donor)
 Liver (Deceased-donor and Living-Donor)
 Pancreas (Deceased-donor only)
 (Deceased-donor only)
 Tissues, cells, fluids
 Hand (Deceased-donor only)
 Cornea (Deceased-donor only Skin graft including
Face transplant (almost always autograft)
 Islets of Langerhans (Pancreas Islet Cells)
(Deceased-donor and Living-Donor)
 Bone marrow/Adult stem cell (Living-Donor and
Autograft)
 Blood transfusion/Blood Parts Transfusion (Living-
Donor and Autograft)
 Blood vessels (Autograft and Deceased-Donor)
 Heart valve (Deceased-Donor, Living-Donor and
Xenograft[Porcine/bovine])
 Bone (Deceased-Donor, Living-Donor, and
Autograft)
 Skin(Deceased-Donor, Living-Donor, and Autograft)90

91. Forensic medicine [DNA finger printing]
 It is a branch of medicine that provides evidences for legal cases such as
 parental testing,
 determination of cause of death,
 identification of criminals from crime scene etc.
 DNA fingerprinting is one of the methods employed in forensic medicine.
 It is the characterization of one or more relatively rare features of an individuals genome or
hereditary make up.
91

92.  The human genome has more or less the same composition among different individuals.
 The same genes will be in the same order.
 However, the genome still shows many polymorphism.
Polymorphism refers to nucleotide sequences that are not found in the same positions
and or in the same order of sequence.
These polymorphic sequences are used to differentiate one from another
92

93. Stem cell research and its potential in medicine
 Research on stem cells is about
 how an organism develops from a single cell and
 how healthy cells replace damaged cells in adult organisms
 leads scientists to investigate the possibility of cell-based therapies to treat disease
 referred as regenerative or reparative medicine.
 Stem cells have two important characteristics that distinguish them from other types of cells
 they are unspecialized cells that renew themselves for long periods through cell division
 under certain physiologic or experimental conditions, they can be induced to become cells with
special functions 93

94. Kinds of Stem Cells
 Unipotent stem cells form only one type of specialized cell type.
 Multipotent stem cell can form multiple types of cell and tissue types
 Pluripotent stem cells can form most or all cell types in the adult.
 Totipotent stem cell can form all adult cell type as well as the specialized tissues to support
development of the embryo (eg. The placenta)
Sources of stem cells
Embryonic stem cells
 are harvested from the inner cell mass of the blastocyst seven to ten days after fertilization.
94

95. Fetal stem cells
 Are taken from the germline tissues that will make up the gonads of aborted fetuses.
Umbilical cord stem cells
 Umbilical cord blood contains stem cells similar to those found in bone marrow.
Placenta derived stem cells
 up to ten times as many stem cells can be harvested from a placenta as from cord blood.
Adult stem cells
 Many adult tissues contain stem cells that can be isolated.
95

96. Stages of Embryogenesis
96
Day 1
Fertilized egg
Day 2
2-cell embryo
Day 3-4
Multi-cell embryo
Day 5-6
BlastocystDay 11-14
Tissue Differentiation
Isolate inner cell mass
(destroys embryo)
Heart muscleKidney
Liver
“Special sauce”
(largely unknown)
Day 5-6
Blastocyst
Inner cells
(forms fetus)
Outer cells
(forms placenta)
Heart
repaired
Culture cells
Derivation and Use of Embryonic Stem Cell Lines

97. Possible Uses of Stem Cell Technology
Replaceable tissues/organs
Repair of defective cell types
Delivery of genetic therapies
Delivery chemotherapeutic agents
97