For MBBS, BDS and MD students

1. netic enGineerinG
Prof. (Dr.) V. P.
Acharya

2. Genetic EngineeringGenetic Engineering
Synthesis and repair of the genes of livingSynthesis and repair of the genes of living
thingsthings
Possible to synthesise genes in the labPossible to synthesise genes in the lab
Paul Berg Herbert Boyer Stanley N. Cohen

3. Recombinant DNARecombinant DNA
Hybrid combination of 2 DNA fragments,Hybrid combination of 2 DNA fragments,
derived from 2 different sourcesderived from 2 different sources
In vivoIn vivo also takes placealso takes place
Genetic recombinationGenetic recombination– normal biological– normal biological
exchange or addition of genes to form anexchange or addition of genes to form an
altered chromosome that can bealtered chromosome that can be
replicated, transcribed and translatedreplicated, transcribed and translated
Eukaryotes– in germ cell conjugationEukaryotes– in germ cell conjugation

4. Artificial recombinant DNAArtificial recombinant DNA
In vitroIn vitro genes are recombinedgenes are recombined
Isolation and manipulation of DNA toIsolation and manipulation of DNA to
make chimeric or hybrid DNAmake chimeric or hybrid DNA
 Chimeric DNA-- molecules containingChimeric DNA-- molecules containing
both human and bacteria DNA sequencesboth human and bacteria DNA sequences

5. CHIMERA

6. The technologyThe technology
 DNA is purified from cells or tissuesDNA is purified from cells or tissues
 Enzymes are used to cut DNA moleculesEnzymes are used to cut DNA molecules
and join them togetherand join them together
 DNA is transferred to a host cellDNA is transferred to a host cell
((transformationtransformation))
 DNA replicated in cellsDNA replicated in cells
If appropriate sequences areIf appropriate sequences are
present, gene may be transcribedpresent, gene may be transcribed
and translated to proteinand translated to protein

8. Restriction Endonuclease (RE)Restriction Endonuclease (RE)
Molecular scissorsMolecular scissors
Selectively gene is split from parent DNASelectively gene is split from parent DNA

10. Restriction sitesRestriction sites
Specific recognition sites where restrictionSpecific recognition sites where restriction
endonucleases actendonucleases act
Recognise sequences withRecognise sequences with palindromepalindrome
sequences/sequences/ invert repeat sequencesinvert repeat sequences
REs produceREs produce sticky ends / cohesive endssticky ends / cohesive ends
or blunt endsor blunt ends

12. VectorsVectors
 To transfer human gene to bacteria , aTo transfer human gene to bacteria , a
carrier required– vectorcarrier required– vector
 PlasmidPlasmid– circular double-stranded DNA– circular double-stranded DNA
in bacteriain bacteria
 CosmidCosmid– plasmid that can carry 20-50– plasmid that can carry 20-50
kbp long DNA; plasmids into which DNAkbp long DNA; plasmids into which DNA
sequences from bacteriophage has beensequences from bacteriophage has been
insertedinserted
 BacteriophageBacteriophage– Carries 10-20 kbp long– Carries 10-20 kbp long
DNA; viral DNA capable of replicating inDNA; viral DNA capable of replicating in
bacterial cell.bacterial cell.

15. Cloning of chimeric moleculeCloning of chimeric molecule
Cloning– Production of large no. ofCloning– Production of large no. of
identical DNA moleculesidentical DNA molecules
Hybrid molecules are amplified by cloningHybrid molecules are amplified by cloning
techniquetechnique

16. Transfection of vector into hostTransfection of vector into host
Host E.coli cells and plasmid vectors areHost E.coli cells and plasmid vectors are
incubated in hypertonic mediumincubated in hypertonic medium
containing calcium ionscontaining calcium ions
Proper growth medium suppliedProper growth medium supplied
Only 5% bacterial colonies contain theOnly 5% bacterial colonies contain the
desired vectordesired vector

17. Recombinant DNAs are propagated asRecombinant DNAs are propagated as
clones in each colony of bacteria.clones in each colony of bacteria.

18. Selection of desired coloniesSelection of desired colonies
Based on the property they have acquiredBased on the property they have acquired
due to the foreign DNAdue to the foreign DNA
Selected colonies again clonedSelected colonies again cloned

19. Applications of recombinant DNA technologyApplications of recombinant DNA technology
 Recombinant Vaccines, hormonesRecombinant Vaccines, hormones
 Interferon, Interleukins, growth factorsInterferon, Interleukins, growth factors
 Monoclonal antibodiesMonoclonal antibodies
 Cell/tissue cultureCell/tissue culture
 Production of next generation antibioticsProduction of next generation antibiotics
 ForensicsForensics
 ErythropoietinErythropoietin
 Blood factorsBlood factors
 Genetically modified cropsGenetically modified crops
 Gene therapyGene therapy
 Diagnosis of diseasesDiagnosis of diseases

20. DNA Library

21. Collection of cloned restriction fragmentsCollection of cloned restriction fragments
of DNA that represents the entire genomeof DNA that represents the entire genome
Also calledAlso called SHOTGUN COLLECTIONSHOTGUN COLLECTION
Genomic DNA library– both introns andGenomic DNA library– both introns and
exons are representedexons are represented
Complementary DNA library– only exonsComplementary DNA library– only exons
are representedare represented

24. Human Genome projectHuman Genome project
US dept of Energy and NIH– 1990US dept of Energy and NIH– 1990
Scientists from 16 centers all over theScientists from 16 centers all over the
worldworld
Whole human genome was decoded andWhole human genome was decoded and
sequencedsequenced
6 million bp per cell and 10,000 genes6 million bp per cell and 10,000 genes
Completed by 1994Completed by 1994
The Book of Human Life–The Book of Human Life– 23 chapters23 chapters

26. For more ppt on medical BiochemistryFor more ppt on medical Biochemistry
Please visitPlease visit
www.vpacharya.comwww.vpacharya.com