I have prepared this lecture presentation on the topic Genetic Engineering for our students and also delivered guest lecture on this topic at various colleges in our region.

1. GENETIC ENGINEERING
Dr. Mangesh J Dagawal
( M.Sc. M.Phil. Ph.D. NET )
Assistant Professor & Head
Department of Botany
Smt. Radhabai Sarda College , Anjangaon, Surji
Dist. Amravati (MS) India
mdagawal@gmail.com
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2. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 2

3. GENETIC ENGINEERING
• The manipulation of genetic material to
produce specific results in an organism.
• The manipulation of a living genome by
introducing or eliminating specific genes
through recombinant DNA techniques,
which may result in a new capability.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 3

4. • This technique is used to produce new
genetic combinations that are of value to
medicine, agriculture, or industry.
• Through recombinant-DNA techniques,
bacteria have been created that are capable
of synthesizing human insulin, human
interferon, human growth hormone, a
hepatitis-B vaccine, and other medically
useful substances.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 4

5. Recombinant DNA technology
• Recombinant DNA molecule is produced by
joining two or more DNA segment usually
originating from different organism.
• r-DNA is a vector in to which desired DNA
fragment has been inserted for cloning in host.
• Gene or DNA cloning produces large number of
copies of gene cloned term- r DNA technology.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji.
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6. STEPS IN GENE CLONING
• Production and isolation of DNA fragment.
• Insertion of isolated gene in vector
• Introduction in to suitable host ( transformation)
• Selection of transformed host cells.
• Multiplication / expression of introduced gene in
host.
• Transfer gene in to other organism.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 6

7. TOOLS AND TECHNIQUE
• Recombinant DNA Technolgy
Tools-
• Enzymes
 Molecular Scissor ( Restriction Endonuclease)
 Molecular Glue ( DNA ligase)
• Vectors
Techniques-
 PCR ( Gene Amplification)
 DNA Sequencing
 Gene Transfer- Direct/Indirect
 DNA Fingerprinting
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 7

8. RESTRICTION ENDONUCLEASES
• A class of endoncleases cleaves DNA only within
or near those site which have specific base
sequences.
• Site recognized by enzyme called recognition
sequence or site.
• Recognition sequence-
• Restriction site :
5’ GAA TTC 3’ Recognition sequence
3’ CTT AAG 5’
Restriction site
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College,
Anjangaon Surji.
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9. NOMENCLATURE
• First letter of the name genus in which given
enzyme discovered written in capital.
• Followed by first two letter of species name of
organism. Letters written in italics.
• Strain or type identification as subscript.
• When organism produces more than one enzyme
identified by roman numeral.
• All restriction enzyme designated by symbol R.
• EcoRI & HindII
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College,
Anjangaon Surji. 9

10. CLEAVAGE PATTERN
Staggered Cut
5’------ GAATTC ---- 3’
3’-------CTTAAG -----5’
5’----G AATTC -- 3’
3’----CTTAA G-- 5’
Protruding end
5’ ----- AGCT-----3’ 5’-------- AG CT------3’
3’------TCGA-----5’ 3’---------TC GA-----5’
Blunt ends
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 10

11. Type II- Restriction endonucleases
• Stable & induces cleave either within or
outside their recognition sequence.
• 350 type II With 100 Different Recognition
Sequence.
• Used for restriction mapping and gene
cloning.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 11

12. VECTORS
• DNA molecule that has ability to replicate in an
appropriate host cell and into which the DNA
fragment to be cloned.
• Properties-
• It should be able to replicate autonomously.
• Easy to isolate and purify
• Easily introduced in host cell.
• Suitable marker genes.
• Unique target site.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 12

13. VECTORS TYPES
• Plasmid
• Phase
• Cosmid
• Phasmid
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 13

14. PLASMID
• Plasmids are extrachromosomal self replication
double stranded closed circular DNA.
• Vary in size from 1kb to 250 kb.
• Minimum amount of DNA (Less than 10kb to
avoid problem)
• At least two selectable marker
• Types-
• F- Plasmid (Conjugation)
• R- Plasmid ( Resistance to antibiotics)
• Col- Plasmid (col- colicin kill sensitive cells.)
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 14

15. Type pBR322
p- signifies- plasmid
• B is for Boliver and R for Rodriguez.
• Numeral 322 denotes this plasmid from other plasmid
developed in same laboratory.
• Size 4363 bp.
• Two selectable marker ( tetracycline tet & ampicillin
amp resistance gene)
• Single recognition site for 12 different restriction enzyme
within selectable marker.
• (Pst I within amp and Bam HI within tet)
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 15

16. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji.
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Dig source google.com

17. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji.
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Dig source google.com

18. PHASE
• Bacteriophase- virus that attack bacteria .
• Several bacteriophase are used as cloning vector.
• Lambda and M13.
• Phase have two advantage over plasmid-
• They are more efficient than plasmid for cloning large
DNA fragment.
• Largest cloned insert size over 25 kb
• Easier to screen large no of phase plaques than bacterial
colonies for identification of recombinant vector.
• Linear DNA.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 18

19. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji.
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20. COSMID
• Cosmid defined as hybrid vector derived from
Plasmid- which contain cos site of phase .
• Plasmid contain minimum of 250 bp of lambda DNA
• COS site ( Sequence yielding cohesive end)
• Sequences needed for binding & cleavage.
• Cosmid has- replication origin, restriction site,
selectable marker from plasmid.
• Use to clone DNA insert up to 40 kb.
• Cosmid are attractive for construction of genomic
library of eukaryotes since they can be used for
cloning large fragment.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 20

21. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 21

22. GENE SOURCE
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji.
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Dr. Mangesh J Dagawal
( M.Sc. M.Phil. Ph.D. NET)
Assistant Professor & Head
Department of Botany
Smt. Radhabai Sarda College , Anjangaon, Surji
Dist. Amravati
mdagawal@gmail.com

23. GENOMIC LIBRARY
• Mixture of clones which when derived directly from
genomic DNA called genomic DNA library.
• Contains at least one copy of every DNA sequence in
the genome.
• Prepared by using restriction endonuclease .
• Cloning of entire genome in the form of library of
random genomic clones shotgun expt.
• Fragment of varying sizes having cut at different places
of genome lead to cut at inconvinient places so fragment
having complete gene will difficult to obtain.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji.
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24. Construction genomic library
• Total genomic DNA extracted.
• Extracted DNA broken in to fragment use in restriction
enzyme.
• R.E. having 4 base and 6 base. recognition sequence.
• 4 base sequence occur 44= 256 and 6 base- 66= 4096
• Single or mixed digestion.
• Partially digested DNA subjected to DNA electrophoresis
Separation of fragment.
• Fragment then inserted in to vector.
• Vector cloned in to bacterial host.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda
College, Anjangaon Surji.
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25. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 25
Dig source google.com

26. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 26
Dig source google.com

27. c – DNA Library
• c DNA is copy or complementary DNA produced by
using mRNA as template.
• DNA copy of an RNA molecule is produced by the
reverse transcriptase .
• When eukaryotic m RNA is used as template poly T
oligonucleotide used as primer.
• Appropriate oligonucleotide primer ( oligo -T) is
annealed with m RNA . Primer base pair to 3’ end of m
RNA .
• Reverse transcriptase extends 3’ end of primer using m
RNA molecule as template.
• Produces RNA-DNA hybrid .
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 27

28. • RNA strand digested either by RNase H or alkaline
hydrolysis.
• End of c DNA serves as its own primer & provide free 3’
OH for synthesis of complementary strand.
• Short hairpin loop is generated at this end.
• Loop cleaved by single strand specific nuclease.
• c-DNA library is a population of bacterial transformants
or phase lysates in which each m RNA isolated from an
organism as its c DNA insertion in plasmid or phase
vector.
• Frequency of c DNA in library depend on frequency of
m RNA.
Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 28

29. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda College, Anjangaon Surji. 29
Steps in c–DNA formation

30. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda
College, Anjangaon Surji.
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Dig source google.com

31. Dr. Mangesh J Dagwal, Smt. Radhabai Sarda
College, Anjangaon Surji.
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Remaining topic in next ppt - Gene Transfer