SKV rDNA Technology & Hybridoma Technology

The content is about the general description of genetic material and further two techniques of biotechnology. The content includes two topics.
Firstly with introduction to biotechnology it describe about DNA, recombinant DNA (rDNA) technology, history, goals, procedure of rDNA technology, tools, techniques, application, demerits and products of rDNA technology.
Second portion entitiled as Hybridoma technology. this includes the basic principle, production of monoclonal antibodies, merits demerits and drugs from monoclonal antibody.
New pharmaceuticals derived from biotechnology is covered in last. All the content is referred from books and internet sources.

1. RECOMBINANT DNA
TECHNOLOGY
&
HYBRIDOMA TECHNOLOGY
Presented To Ms. GulnazFatima
By Sachin kumar Vishwakarma
-M.Pharm(Pharmaceutical Chemistry)
1

2. BIOTECHNOLOGY
■Biotechnology is now defined as “Any technological application that
uses biological systems, living organisms or derivatives thereof to
make or modify products or processes for specific uses”.
■Biotechnology includes following technologies :-
1.Bioprocessing Technology
2.Cell Culture
3.Recombinant DNA Technology
4.Cloning
5.Protein Engineering
6.Biosensors
7.Nano-biotechnology
2

3. DNA (DEOXYRIBOSE NUCLIC ACID)
■Nucleic acid was first isolated by Johann Friedrich Miescherin 1868 from the pus
cells. He called it as “nuclein” because of its acidic nature.
■DNA is a double-stranded molecule, made up of the two chains of nucleotides.
■Each nucleotide of DNA consists of three parts:-
a.Nitrogenous Base (adenine, guanine, thymine, cytosine )
b.Pentose Sugar
c.Phosphate group
■In 1953, F.H.C. Crickof Great Britain and J.D. Watson, a young American scientist,
published a brief paper describing how these three components might be arranged
in DNA.
■In the Watson–Crick double helix model, DNA resembles a spiral ladder.
■The functions of DNA is carrying huge amounts of information that determines all
biological activities of an organism, and which is transmitted from one generation
to the next.
3

4. 4

5. RECOMBINANT DNA (rDNA) TECHNOLOGY
■RecombinantDNAtechnology,whichisalsocalledgenecloningisan
umbrellatermthatencompassesanumberofexperimentsprotocols
leadingtothetransferofgeneticinformation(DNA)fromoneorganismto
another.
OR
DNAmoleculesthatareextractedfromdifferentsourcesandchemically
joinedtogether;forexampleDNAcomprisingananimalgenemaybe
recombinedwithDNAfromabacterium.
■RecombinationofDNAoccursnaturallyinmicrobes.Inthe1970sand
1980s,scientistsdevelopedartificialtechniquesformakingrecombinant
DNA
.
5

6. Discovery of rDNA technology
■DiscoveryofDNAstructureWatson&Crickin1953
■IsolationofDNAligasein1967
■IsolationofREasein1970
■PaulBerggeneratedrDNAtechnologyin1972
■Cohen&Boyerin1973producedfirstplasmidvector(Intheir
experiments,theysuccessfullyrecombinedtwoplasmids(pSC101
andpSC102)andclonedthenewplasmidinE.coli.)
■capableofbeingreplicatedwithinabacterialhost
6

7. Goals of recombinant DNA technology
■
To isolate and characterize a gene
■To make desired alterations in one or more isolated genes
■To return altered genes to living cells
■Artificially synthesize new gene
■Alternating the genome of an organism
■Understanding the hereditary diseases and their cure
■Improving human genome
7

8. Procedure of making rDNA
Following 4 steps are involved in process of recombinant DNA technology:-
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9. Isolation of DNA
9

10. Cutting of DNA
■DNAcanbecutintolargefragmentsby
mechanicalshearing.
■Restrictionenzymesarethescissorsof
moleculargeneticsandusedtocutthe
DNAinaspecificsequenceandso
calledassequence-specificenzyme.
■Sequence-specificenzymearefoundin
bacteria.
■Restrictionendonucleases(REases)
recognizeDNAbasesequencethatare
palindrome.
■REasemakestaggeredcutswith
complementarybasesequencesfor
easycirculization.
10

11. Joining DNA
11

12. Amplifying the recombinant DNA
■TransformingtherecombinantDNAintoabacterialhoststrain.
■ThecellsaretreatedwithCaCl2
■DNAisadded
■Cellsareheatshockedat42C
■DNAgoesintocellbyasomewhatunknownmechanism.
■Onceinacell,therecombinantDNAwillbereplicated.
■Whenthecelldivides,thereplicatedrecombinantmoleculesgotoboth
daughtercellswhichthemselveswilldividelater.Thus,theDNAis
amplified.
12

13. Amplifying the recombinant DNA(Cont.)
13

14. rDNA TECHNOLOGY (Complete process)
14

15. 15

16. TOOLS OF rDNA TECHNOLOGY
Following tools are used in recombinant DNA technology:-
1.Restriction endonucleases
2.DNA ligases
3.Vectors
4.Others
a.Mutagens (physical and chemical agents hat produce mutations)
b.Use of Reverse Transcriptase to synthesize DNA
c.Synthetic nucleic acids
16

17. Restriction endonucleases
■BacterialenzymesthatcutDNAmoleculesonlyatrestrictionsites.
■Bacterialorigin=enzymesthatcleaveforeignDNA
■Namedaftertheorganismfromwhichtheywerederived;
Examples:-EcoRI is from Escherichia (E) coli (co), strain Ry13 (R), and first
endonuclease (I) to be discovered.
■Protectbacteriafrombacteriophageinfection(Restrictsviralreplication).
■TypeI–CutstheDNAonbothstrandsbutatanon-specificlocationatvarying
distancesfromtheparticularsequencethatisrecognizedbytherestriction
enzyme.
■TypeII–CutsbothstrandsofDNAwithintheparticularsequencerecognized
bytherestrictionenzyme.
17

18. DNA ligases (DNA joining enzymes)
■The cut DNA fragments are covalently joined together by DNA ligases.
■These enzymes were originally isolated from viruses. They also occur in E.coli and
eukaryotic cells. DNA ligases actively participate in cellular DNA repair process.
■DNA ligase joins (seals) the DNA fragments by forming a phosphodiester bond between
the phosphate group of 5’-carbon of one deoxyribose with the hydroxyl group of 3’-carbon
of another deoxyribose.
18

19. Vectors (Carriers)
In genetic engineering, the vector (carrier) is the most widely used
method for the insertion of foreign, or passenger, genetic material into a
cell.
Types:-
1.Plasmid vectors
2.Lamda(λ) phase vectors
3.Cosmidvectors
4.Expression vectors
5.Yeast Artificial Chromosomes (YACS)
6.Bacterial Artificial Chromosomes (BACS)
19

20. Plasmid Vectors
■Plasmidsareextrachromosomal,doublestranded,
circular,self-replicatingDNAmolecules.
■Theyreplicateindependentlyofthebacterial
chromosome.
■UsefulforcloningDNAinsertslessthat20kb
(kilobasepairs).
■Insertslargerthan20kbarelosteasilyinthe
bacterialcell.
■Nomenclatureofplasmids:-Itisacommon
practicetodesignateplasmidbyalowercasep,
followedbythefirstletter(s)ofresearcher(s)
namesandthenumericalnumbergivenby
theworkers.
■Example:-pUC19isplasmidfromUniversityof
Californiaandresearchersdesignatedas19.
20

21. Techniques used in rDNA technology
Following techniques are used in rDNA technologies:-
■PCR (Polymerase Chain Reaction )
■Gel electrophoresis
■Cloning libraries
■Restriction enzyme mapping
■Nucleic Acid Hybridization
■DNA Microarrays
21

22. PCR (Polymerase Chain Reaction)
■Thepolymerasechainreaction(PCR)isatechniquebywhichsmall
samplesofDNAcanbequicklyamplified,thatis,increasedto
quantitiesthatarelargeenoughforanalysis.
■Startingwithjustonegene-sizedpieceofDNA,PCRcanbeusedto
makeliterallybillionsofcopiesinonlyafewhours.
■EachstrandofthetargetDNAwillserveasatemplateforDNA
synthesis.
■Shortpiecesofnucleicacidcalledprimersarealsoaddedtohelp
startthereaction.
■TheprimersarecomplementarytotheendsofthetargetDNA.
■Aftereachcycleofsynthesis,theDNAisheatedtoconvertallthenew
DNAintsinglestrands.EachnewlysynthesizedDNAstrandservesin
turnasatemplateformorenewDNA.
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23. Polymerase Chain Reaction
23

24. Applications of rDNA technology
■Agriculture:growingcropsofyourchoice(GMfood),pesticideresistant
crops,fruitswithattractivecolors,allbeinggrowninartificialconditions.
■Pharmacology:artificialinsulinproduction,drugdeliverytotargetsites.
■Medicine:genetherapy,antiviraltherapy,vaccination,synthesizing
clottingfactors.
■Otheruses:-fluorescentfishes,glowingplantsetc.
24

25. DEMERITS Of rDNA TECHNOLOGY
■Highcostprocess
■Requiresophisticatedenvironment
■Morestepsforasingleproducts(eg.:-processingoftherecombinant
protein)
■Chemicalinstabilityofproducts(hydrolysis,deamidation,
racemization,oxidation)
■Physicalinstabilityofproteins(denaturation,adsorption,aggregation,
precipitation)
■immunogenicity
25

26. DRUG DISCOVERY rDNA
TECHNOLOGY
■Oneofthemostmedicallysignificantapplicationsof
recombinantDNAtechnologyisthemodificationofbacterial
cellstomakesubstancesusefultohumans.
■Tomakebacterialcellsproducehumanproteins,ahuman
DNAgenewiththeinformationforsynthesizingtheproteinis
insertedintothevector.
26

27. RECOMBINANT DNA PRODUCTS
A.Hormones
B.Cytokines
C.Interferons
D.Interleukins
E.Enzymes
F.Vaccines
27

28. A. Hormones
a.Humaninsulin:-Humaninsulinwasthefirstpharmacologicallyactive
biologicalmacromoleculetobeproducedthroughgenetic
engineering.TheFDAapprovedthedrugin1982fortreatmentof
type1(insulin-dependent)diabetes.
Theinsulinproteinisatwo-chainpolypeptidecontaining51amino
acidresidues.ChainAiscomposedof21aminoacids,andchainB
contains30.
Brandnames:-HUMUIIN,NOVOLIN
b.Glucagon,
c.Humangrowthhormone,
d.Follicle-stimulatinghormone
28

29. Production
of
recombinant
insulin
29

30. B. Cytokines
a.Erythropoietin Alfa:-It is a glycoprotein that stimulates red blood cell
production. Epoetinalfa(EPOGEN) is a 165 –amino acids
glycoprotein that is manufactured in mammalian cells by rDNA
technology.
Brand names:-EPOGEN, PROCRIT, EPOETIN ALFA
Epoetinis indicated to treat anemia.
b.Filgrastim
c.Sargramostim
d.Becapermin
30

31. C. Interferons
The interferons are family of small proteins or glycoproteins of
molecular masses ranging from 15,000 to 25,000 Da and 145 to 166
amino acids long.
Their action is bimodal. The immediate effect is the recruitment of
natural killer cells to kill the host cell harbouring the virus. Interferons
then induce a viral resistance in cell in the immediate vicinity,
preventing spread of the virus.
a.Interferon Alfa-2a (recombinant)
b.Interferon Alfa-2b (recombinant)
c.Interferon Beta-2a (recombinant)
d.Interferon Beta-2b (recombinant)
31

32. D. Interleukins
a.Aldeslukin
b.Oprelvekin
c.Tumornecrotic Factor (recombinant):-The TNFs are members of a
family cytokinine.sthat are produced primarily in the innate immunity
system by activated mononuclear phagocytes.
Brand name:-ETANERCEPT, ENBREL
32

33. E. Enzymes
a.Blood-Clotting Factors:-
•Tissue Plasminogen Activator
•Reteplase
•Tenecteplase
•Factor VIII
b.Anticoagulants:-
•Lepirudinrecombinant:-It is an rDNA-derived protein produced in
yeast, has a molecular mass of approximately 7,000 Da.
33

34. F. Vaccines
Vaccine production is a natural application of rDNA technology, aimed
at achieving highly pure and efficacious products.
Current, there are four rDNA vaccines approved for human use
.
a.Recombivaxand Engerix-B:-For immunization against hepatitis B
virus. Both contain a 226-amino acid polypeptide chain.
b.LYMErix
c.Comvax:-It is a combination of Haemophilus influenzatype b
conjugate and hepatitis B (recombinant).
34

35. HYBRIDOMA
TECHNOLOGY
35

36. HYBRIDOMA TECHNOLOGY
■AhybridomaisahybridcellobtainedbyfusionofBlymphocytewith
usuallyatumorcellofantibodyformingsystemorBlymphocyte
(thesearecalledmyelomas).
■In1975,GeorgeKohlerandCesarMilstein(NobelPrize1984)made
thisdreamareality.Theycreatedhybridcellsthatwillmakeunlimited
quantitiesofantibodieswithdefinedspecificities,whicharetermed
asmonoclonalantibodies(McAb).Thisdiscovery,oftenreferredtoas
hybridomatechnology,hasrevolutionizedmethodsforantibody
production.
■ThetermhybridomawascoinedbyLeonardHerzenbergin1975.
36

37. PRINCIPLE
■Thisisbasedonthe fusionbetweenmyelomacells(malignantplasma
cells)andspleencellsfromasuitablyimmunizedanimal.Spleencellsdie
inashortperiodunderordinarytissuecultureconditionswhilemyeloma
cellsareadoptedtogrowpermanentlyinculture.
■Fromthegrowinghybrids,individualclonescanbechosenthatsecrete
thedesiredantibodies(monoclonalorigin).Theselectedcloneslike
ordinarymyelomacellscanbemaintainedindefinitely.
37

38. MONOCLONAL ANTIBODY
DEVELOPMENT PROCESS
■Immunization of appropriate animals with antigen (need not be pure)
under study.
■Fusion of suitable drug resistant myeloma cells with plasma cells,
obtained from the spleen of the immunized animal.
■Selection and cloning of the hybrid cells that grow in culture and
produce antibody molecules of desired class and specificity against
the antigen of interest.
38

39. 39
Production of Monoclonal
Antibodies

40. MERITS OF HYBRIDOMA TECHNOLOGY
■Hybridomatechnologycanmakeavailablehighlyspecificantibodiesin
abundantamounts.
■Theclonesoncedevelopedarefarcheaperthanthetraditionally
employedanimals
■(horses,rabbits)forproducingantibodies.
■Theclonesdevelopedfromthehybridswillalsoensureconstancyofthe
qualityoftheproductandwillalsoavoidthebatchtobatchvariation
inherentintheconventionalmethods.
40

41. DEMIRITS OF HYBRIDOMA TECHNOLOGY
■Many patients develop immune response to monoclonal antibodies
produced by mice, as these are foreign proteins.
■Hybridomaculture may be subjected to contamination.
■System is only well developed for mouse and rat and not for the other
animals.
■More than 99% of the cells do not survive during the fusion process-
reducing the range of useful antibodies that can be produced against
an antigen.
41

42. APPLICATIONS OF HYBRIDOMA
TECHNOLOGY
1.ProductionofMonoclonalantibodies
2.EarlydetectionofPregnancy
3.DiagnosisofHIV(detectionofpresenceofHIVantibody)
4.IdentificationoftypesofLeukaemias(Monoclonalantibodiescan
distinguishsubsetsofBcellsandTcells)
5.DiagnosisandtreatmentofCancer
42

43. MONOCLONAL ANTIBODY DRUGS
RITUXIMAB
GEMTUZUMAB OZOGAMICIN
BASILIXIMAB
DACLIZUMAB
ABCIXIMAB
INFLXIMAB
ALEMTUZUMAB
43

44. NEW PHARMACEUTICALS
DERIVED FROM
BIOTECHNOLOGY
44

45. INTRODUCTION
■Biotechnologyencompassesanytechniquethatuseslivingorganisms
intheproductionormodificationofproducts.
■Thus,biotechnologyproductsbroadlyrefertobiopharmaceuticaldrugs
generatedthroughresearchesincellbiology,geneticsand
recombinantDNAtechnology.
45

46. PHARMACEUTICALS
DERIVED FROM
BIOTECHNOLOGY
46

47. A. rDNA Technology
a.Hormones
b.Cytokines
c.Interferons
d.Interleukins
e.Enzymes
f.Vaccines
47

48. B. Hybridomatechnology
a.Antibody production
b.Diagnosis of diseases (ELISA, RIA)
c.Treatment of various diseases
d.Development of various drugs
48

49. C. Gene therapy
a.Treatment of Diseases on Genetic level
b.Modification in hereditary characteristic
c.Genetically modified plants (increase drug amount/potency )
d.Genetically modified animals (biological products and organs)
e.Transgenic animals
49

50. D. Fermentation
a.Production of wine
b.Production of Antibiotics
50

51. PRODUCTS OF BIOECHNOLOGY

Anticoagulantdrug– Lepirudin(Refludan
®
)

Antisensedrugs –Fomivirsensodiuminjection
isapproved
forlocaltreatmentofcytomegalovirus
(CVM)retinitisinpatientswithAIDS

Efavirenz(Sustiva
®
)–Anon-nucleoside
reversetranscriptaseinhibitorand
thefirstanti-HIV
drugtobeapprovedbyFDAforoncedailydosingin
combinationwithotheranti-HIVdrugs.
51

52. PRODUCTS OF BIOECHNOLOGY (Cont.)

Clotting factors-Kogenate
®
, Recombinate
®
) –
Recombinant anti-hemophiliac factor
indicated for
the treatment of classical hemophilia A in which there is a
demonstrated deficiency of clotting factor (Factor VIII )

Growth factor–Becaplemin(regranex
®
)

Systemic growth hormone - Somatropinrecombinant
(Humatrope®)
52

53. PRODUCTS OF BIOECHNOLOGY (Cont.)

Colony stimulating factors -

Erythropoietin

Interferon

Vaccines –Genetically engineered vaccines use a
synthetic copy of the protein coat of a virus to “fool” the
body immune system into mounting a protective
response.
53

54. REFERENCES
1.Tortora,G.J.;Funke,B.R.,“Microbiology”;10
th
Edition;BenjaminCummings;
SanFrancisco.
2.Satyanaryana,U.;Chakrapani,U.;“Biochemistry”;Elsevier,NewDelhi.
3.Black,J.G.;”Microbiology”;8
th
Edition;JohnWileyandSons.
4.Willey,J.M.;Sherweood,L.M.;“Prescott,Harley,andKlein’sMicrobiology”;
7
th
Edition;McGrawHill,Boston.
5.Beale,J.M.;Block,J.H.;“WilsonandGisvold’sTextbookofOrganic
MedicinalandPharmaceuticalChemistry”;12
th
Edition;Lippincott
Williams&Wilkins,NewDelhi.
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55. THANK YOU FOR YOUR ATTENTION
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