The 39 base-pair Epidermal Growth Factor Receptor Antisense Sequence (EGFR AS) s
ID: 3166083 • Letter: T
Question
The 39 base-pair Epidermal Growth Factor Receptor Antisense Sequence (EGFR AS) spans the translation start site for the EGFR gene 5'-CCG GCC GTC CCG GAG GGT CGC ATC GCT GCT CCC CGA AGA-3' Squamous cell carcinoma of the head and neck (SCCHN) is characterized by upregulation of the EGFR. The antisense sequence described above represents the first human application of down regulating EGFR expression producing anti-cancer activity (JCO 2009, 27, 1235). (a) Plasmid vectors may.be used as delivery vehicles to internalize DNA into cells for cloning and replication. Describe how you would construct the plasmid vector indicated below for cloning multiple copies of the EGFR AS gene. Note: KanR refers to antibiotic resistance gene, U6 is the promoter site, ori is the origin of replication and polyA refers to polyadenylation US EGFR AS poly A KanR ori pNGVL-1-U6-EGFRASExplanation / Answer
This plasmid vector can be genetically engineered to construct the same and obtain the desired product. Here, a U6 promoter can be added to a pre-defined site on the plasmid of the host along with polyA site from the target. This polyA site will help in finding the termination site for the siRNA against EGFR. The ori site can be added to the initiation site of the plasmid for transcription followed by kanamycin resistance gene to act as a selectable marker site on the plasmid.
These genetic sequences can be digested with appropriate restriction endonuclease enzymes to give sticky ends and then sealed with enzyme ligase in the presence of a phosphpate donor. This will be followed by action of enzyme alkaline phosphatase to seal the ends and give appropriate binding sites at the 5' and 3' ends of the linear sequences. Finally, circularization of plasmid is performed in the presence of enzyme ligase.
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