This document summarizes various tools used in genetic engineering. It describes restriction enzymes like EcoRI and HinDIII that cut DNA at specific sequences. It also discusses DNA ligases that join DNA fragments and alkaline phosphatase which removes phosphates to prevent unwanted ligation. Common vectors like plasmids, bacteriophages, cosmids, BACs and YACs are outlined which allow insertion of exogenous DNA for storage and manipulation. Larger fragments can be accommodated by vectors like YACs and BACs.

1. TOOLS USED IN GENETIC ENGINEERING
Genes under engineering

2. GENETIC ENGINEERING
 The simple addition, deletion, or
manipulation of a single trait in an
organism to create a desired change.
 Artificially copying a piece of DNA from
one organism and joining this copy of DNA
into the DNA of another organism

3. ENZYMES
• RESTRICTION ENDONUCLEASES
• DNA LIGASES
• ALKALINE PHOSPHATASE
• POLYMERASES

4. Restriction Enzymes
 Enzymes that recognize a specific base
sequence in DNA and cleave at that site
 Isolated from bacteria that leaves
phosphate group on 5’ end & OH group
on the 3’ end
 “Molecular scissors”

5. Nomenclature
EcoRI
• E = Escherichia genus name
• co = coli species name
• R = strain RY12 strain or serotype
• I = Roman numeral one = first enzyme
HinDIII
• Haemophilus influenza serotype d
3rd enzyme

6. TYPES OF RESTRICTION
ENDONUCLEASES

7. TYPES OF CLEVAGES

9. DNA LIGASES
• Ligation is a process of joining nicked
single stranded DNA by the formation
of phosphodiester bond
• Ligation process uses ATP as a co-factor
• Steps involved in ligation
first the co-factors breaks into
AMP which turn acetylate the NH2 group
between the 3’ to 5’ DNA strands

10. Phosphodiester formation - mechanism

11. ALKALINE PHOSPHATASE
• Alkaline phosphatase is an enzyme involved
in the removal phosphate groups
• This enzyme is useful to prevent unwanted
ligation of DNA molecules which is a
frequent problem encountered by cloning
experiments

12. POLYMERASES
• The group of enzymes that catalyse the
synthesis of nucleic acid molecules are
collectively referred to as polymerases
• DNA dependent DNA polymerase that
copies of DNA from RNA
• RNA dependent DNA polymerase (reverse
transcriptase) that synthesis DNA from RNA
• DNA dependent RNA polymerase that
produces RNA from DNA

13. VECTORS
Allowing the exogenous DNA to
be inserted, stored, and
manipulated mainly at DNA level
• Plasmid vectors
• Bacteriophage vectors
• Cosmids
• BACs & YACs

14. plasmid vector
 Plasmids are circular
DNA molecules
present in the
cytoplasm of the
bacteria Capable of
autonomous
replication
 Can transfer genes
from one cell to other
 Act as vectors in
genetic engineering.
 Can also present in
Yeasts

15. Plasmid vectors
Plasmid vectors are double-stranded,
circular, self-replicating, extra-chromosomal DNA
molecules.
• Advantages:
– Small, easy to handle
– Straightforward selection strategies
– Useful for cloning small DNA fragments
(< 10kbp)
• Disadvantages:
– Less useful for cloning large DNA fragments
(> 10kbp)

16. Bacteriophage vectors
Bacteriophages or phages are the viruses that
replicate within the bacteria
Phage vectors can accept short fragments of
foreign DNA into their genome
Bacteriophage
bacteriophage lambda is virus of E.coli, has
been most thoroughly studied and developed as a
vector

17. Phage M13 vectors
Phage M13 (bacteriophage M13) is a single
stranded DNA phage of E.coli
Inside the host cell,M13 synthesizes the
complementary strand to form a double
stranded DNA
If M13 used as a cloning vector replicative
form of DNA is isolated and foreign DNA can
be inserted on it

18. COSMIDS
Cosmids are the vectors possessing the
characteristics of both plasmid and
bacteriophage lambda
Recombinant cosmid is injected into the
bacterial cell where the cosmid arranges into a
circle and replicates as a plasmid. It can be
maintained and recovered just as plasmids.

19. Bacterial artificial chromosome(BAC)
• BACs can hold up to 300 kbs
• The F factor of E.coli is capable of handling
large segments of DNA.
• Recombinant BACs are introduced into E.coli by
electroporation ( a brief high-voltage current).
Once in the cell, the rBAC replicates like an F
factor
Example: Pbac108
• A chloramphenicol resistance gene, and a
cloning segment.

20. Yeast artificial chromosome(YAC)
 YACs can hold up to 500 kbs.
 YACs are designed to replicate as plasmids in
bacteria when no foreign DNA is present. Once a
fragment is inserted, YACs are transferred to cells,
they then replicate as eukaryotic chromosomes.
 DNA is inserted to a unique restriction site, and
cleaves the plasmid with another restriction
endonuclease that removes a fragment of DNA and
causes the YAC to become linear. Once in the cell, the
rYAC replicates as a chromosome, also replicating the
foreign DNA.

21. THANK YOU