Question

From the below list, which of the following is the most logical sequence of steps for splicing foreign DNA into plasmid and inserting the plasmid into a bacterium?
(i) Transform bacteria with recombinant DNA molecule
(ii) Cut the plasmid DNA using restriction enzymes.
(iii) Extract plasmid DNA from bacterial cells
(iv) Hydrogen-bond the plasmid DNA to non plasmid DNA fragments.
(v) Use ligase to seal plasmid DNA to non plasmid DNA
A. (iv), (v), (i), (ii), (iii)
B. (iii), (ii), (iv), (v), (i)
C. (iii), (iv), (v), (i), (ii)
D. (ii), (iii), (v), (iv), (i)

Answer 1

Recombinant DNA technology is the joining together of DNA molecules from two different species. The recombinant DNA molecule is inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.

Complete step by step answer: -Isolation of DNA: The first step of recombinant DNA technology is the isolation of DNA from both vector and donor DNA. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA.
-Cutting of DNA: Restriction enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation.
-Joining of DNA: Both donor DNA and vector DNA are digested with the use of a restriction enzyme that produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form recombinant molecules. When the two populations are mixed, DNA fragments from the two sources can unite, because double helices form between their sticky ends. At this stage, although sticky ends have united to generate a population of chimeric molecules, the sugar-phosphate backbones are still not complete at two positions at each junction. However, the backbones can be sealed by the addition of the enzyme DNA ligase, which creates phosphodiester bonds at the junctions
-Amplifying recombinant DNA: As the donor DNA insert is part of the vector’s length, the donor DNA is automatically replicated along with the vector. Each recombinant plasmid that enters a cell will form multiple copies of itself in that cell. Subsequently, many cycles of cell division will take place, and the recombinant vectors will undergo more rounds of replication.
-The resulting colony of bacteria will contain billions of copies of the single donor DNA insert. This set of amplified copies of the single donor DNA fragment is the DNA clone.
So, the answer is B, i.e., (iii), (ii), (iv), (v), (i)

Note: The ligated recombinant DNA enters a bacterial cell by transformation. After it is in the host cell, the plasmid vector is able to replicate because plasmids normally have a replication origin.