Question

You discovered a novel antibiotic-resistant gene in a bacteria and would like to introduce the gene into a plant. Describe how you would clone and introduce the gene, and how you would determine whether each step was successful.

Answer 1

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

Gene cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. Classic gene cloning involves the following 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 the polymerase chain reaction (PCR) or restriction enzyme digestion.

A restriction enzyme (restriction endonuclease) is an enzyme that cuts double-stranded DNA at a specific sequence. The enzyme makes two incisions, one through each strand of the double helix, without damaging the nitrogenous bases. This produces either overlapping ends (also known as sticky ends) or blunt ends.

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

Ligation

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

The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks. This produces a recombinant DNA molecule.

Figure depicts the producion of recombinant DNA molecule.

Transfection or Transformation

The recombinant DNA is placed into a host cell, usually bacterial, in a process called transfection or transformation. Finally, the transfected cells are cultured. Many of these cultures may not contain a plasmid with the target DNA as the transfection process is not usually 100% successful, so the appropriate cultures with the DNA of interest must be selected.

Selection

Many plasmids/vectors include selectable markers - usually some sort of antibiotic resistance. When cultures are grown in the presence of an antibiotic, only bacteria transfected with the vector containing resistance to that antibiotic should grow.

However, these selection procedures do not guarantee that the DNA of insert is present in the cells. Further analysis of the resulting colonies is required to confirm that cloning was successful.

DNA sequence analysis, PCR, or restriction fragment analysis will all determine the success, i.e. whether the plasmid/vector contains the insert.

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

Gel electrophoresis will separate DNA molecules based on size and charge.

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