TOOLS USED IN GENETIC ENGINEERING

Genes under engineering

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

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

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”

NomenclatureEcoRI

• E = Escherichia genus name

• co = coli species name• R = strain RY12 strain or

serotype• I = Roman numeral one = first

enzymeHinDIII

• Haemophilus influenza serotype d 3rd enzyme

TYPES OF RESTRICTION ENDONUCLEASES

TYPES OF CLEVAGES

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

Phosphodiester formation - mechanism

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

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

VECTORS Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level • Plasmid vectors

• Bacteriophage vectors

• Cosmids

• BACs & YACs

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

Plasmid vectorsPlasmid 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)

Bacteriophage vectorsBacteriophages or phages are the

viruses that replicate within the bacteria

Phage vectors can accept short fragments of foreign DNA into their genome

Bacteriophagebacteriophage lambda is virus of

E.coli, has been most thoroughly studied and developed as a vector

Phage M13 vectorsPhage 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

COSMIDSCosmids 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.

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.

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.

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