You discovered a novel antibio

The process of cloning the novel- antibiotic resistant gene,introducing it into the cell and determining it’s success isdescribed below:

Gene cloning, refers to theprocess of isolating a DNA sequence of interest for the purpose ofmaking multiple copies of it. Classic gene cloning involves thefollowing 4 steps:

1. isolation of the DNA of interest (or target DNA),
2. ligation,
3. transfection (or transformation), and
4. a screening/selection procedure.

Isolation of DNA

IThis DNA of interest may be a gene, part of a gene, a promoter,or another segment of DNA, and is frequently isolated by thepolymerase chain reaction (PCR) orrestriction enzymedigestion.

A restriction enzyme (restrictionendonuclease) is an enzyme that cuts double-stranded DNAat a specific sequence. The enzyme makes two incisions, one througheach strand of the double helix, without damaging the nitrogenousbases. This produces either overlapping ends (also known as stickyends) or blunt ends.

The plasmid or vector is digested withrestriction enzymes, opening up the vector to allow insertion ofthe target DNA. If the isolated DNA of interest and the plasmid orvector are digested with the same restriction enzyme, their stickyends will be complementary.

Ligation

Restriction fragments ( DNA of interest and a plasmid/vector)can be spliced together, provided their sticky ends arecomplementary. Blunt end ligation is also possible.

The two DNAs are then incubated with DNAligase, an enzyme that can attach together strands of DNAwith double strand breaks. This produces arecombinant DNAmolecule.

Figure depicts the producion of recombinant DNAmolecule.

Transfection or Transformation

The recombinant DNA is placed into a hostcell, usually bacterial, in a process calledtransfection or transformation. Finally, the transfected cells arecultured. Many of these cultures may not contain a plasmid with thetarget DNA as the transfection process is not usually 100%successful, so the appropriate cultures with the DNA of interestmust be selected.

Selection

Many plasmids/vectors include selectablemarkers – usually some sort of antibioticresistance. When cultures are grown in the presence of anantibiotic, only bacteria transfected with the vector containingresistance to that antibiotic should grow.

However, these selection procedures do not guarantee that theDNA of insert is present in the cells. Further analysis of theresulting colonies is required to confirm that cloning wassuccessful.

DNA sequenceanalysis, PCR, orrestriction fragmentanalysis will all determine the success, i.e.whether the plasmid/vector contains the insert.

Restriction fragment analysis is digestion of isolatedplasmid/vector DNA with restriction enzymes. If the isolated DNAcontains the target DNA, that fragment will be excised by therestriction enzyme digestion.

Gel electrophoresis will separate DNA molecules based on sizeand charge.

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