Main steps in the formation of Recombinant DNA are given below. Arrange these steps in a correct seq

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Question

Main steps in the formation of Recombinant DNA are given below. Arrange these steps in a correct sequence.
A. Insertion of recombinant DNA into the host cell.
B. Cutting of DNA at specific location by restriction enzyme.
C. Isolation of desired DNA fragment.
D. Amplification of gene of interest using PCR.
Choose the correct answer from the options given below:
(1) C, A, B, D
(2) C, B, D, A
(3) B, D, A, C
(4) B, C, D, A

Accepted Answer

Correct Answer: D — B, C, D, AStep-by-step Solution:The correct sequence for recombinant DNA technology is:Step 1 — B (Cutting of DNA by restriction enzyme): The source DNA (containing the gene of interest) and the vector DNA are both digested with the same restriction endonuclease (e.g., EcoRI). This produces specific DNA fragments with compatible ends (sticky ends or blunt ends).Step 2 — C (Isolation of desired DNA fragment): The restriction-digested DNA fragments are separated by agarose gel electrophoresis. The band corresponding to the gene of interest is identified (by Southern blotting or ethidium bromide staining), excised from the gel, and the DNA is eluted/purified.Step 3 — D (Amplification of gene of interest using PCR): The isolated gene fragment can be amplified by the Polymerase Chain Reaction (PCR) to generate sufficient copies. PCR uses specific oligonucleotide primers flanking the gene, Taq DNA polymerase, and thermocycling (denaturation → annealing → extension).Step 4 — A (Insertion of recombinant DNA into the host cell): The amplified gene is ligated into the vector (e.g., plasmid) using DNA ligase to create the recombinant DNA molecule. This recombinant construct is then introduced into a suitable host cell (e.g., E. coli) by transformation (heat shock/CaCl₂ method) or electroporation. Successful transformants are selected using antibiotic resistance or other selectable markers.Key Concepts:Restriction endonucleases: Molecular scissors; recognise specific palindromic sequences; cut at specific sites; produce sticky or blunt ends. Example: EcoRI recognises 5'-GAATTC-3'.PCR (Polymerase Chain Reaction): In vitro amplification of specific DNA; three steps per cycle: denaturation (94°C) → primer annealing (~55°C) → extension (72°C); exponential amplification.Transformation: Uptake of foreign DNA by a host cell; CaCl₂ treatment makes E. coli competent.Selection of recombinants: Insertional inactivation of antibiotic resistance gene (blue-white colony screening); or antibiotic selection plates.

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