Please answer and explain question 1 o connections As a graduate student you wis

Question

Please answer and explain question 1

o connections As a graduate student you wish to study a specific gene in dogs, the wopay tail lwgt) gene. You previously isolated the gene, and now you want to clone it into a plasmid, called puC19- the map of the plasmid is shown below. The plasmd contains about 2.7 kb (kilobase pairs) of DNA ng: a. Amp b. Ori c. MCS (multiple cloning site) Circle the region on the plasmid where you would clone the wgt gene. Explain your choice. 2. lacz MCS LacR binding site

Explanation / Answer

Ans. #a. Amp: The ampicillin resistance gene (ampR) in plasmid helps the identification of successfully transformed host cell. Generally the antibiotic resistance genes in the plasmid genome works well as the selectable marker. A host cell with recombinant plasmid having a foreign DNA in its ampR gene becomes susceptible to the same antibiotic because of disruption of native gene sequence of the ampR gene. Through replica plate technique, the transformed cell colony (susceptible to amp) can be distinguished from the non-recombinant colony (resistant to amp).

#b. Ori or Origin of Replication: The presence of ‘origin of replication (Ori)’ in the plasmid (cloning vector) is crucial for its autonomous replication inside the suitable host.

#c. Multiple Cloning Sites (MCS): A recognition sequence to which the foreign DNA is ligated into is called a cloning site. Presence of a single recognition sequence of it its type is an ideal cloning site. Presence of two or more number of the same recognition sequence produces multiple fragment of the vector DNA upon specific endonuclease treatment, thus also makes the reconstruction of the functional recombinant vector non-feasible. Two or more (up to ~20) unique (not multiple copies) restriction sites clumped within a short stretch of cloning vector DNA is called multiple cloning site (MCS) or polylinker. MCS acts as the specific target sequence/site in the plasmid genome where gene of interest is to be ligated.

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