Genetic engineering tools and tools based biological functions

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