Q.53 DNA cloning is an essential tool in recombinant DNA technology.
Arrange the different steps of DNA cloning in proper sequential order.

A. Restriction digestion of the insert and plasmid.
B. Selection of the host cells or transformants having the recombinant DNA.
C. In vitro DNA ligation.
D. Transformation of the ligated DNA.
E. Confirmation of recombinant DNA by sequencing.

Choose the correct answer from the options given below:

1. B, C, D, A, E
2. A, D, C, E, B
3. A, C, D, B, E
4. B, A, D, C, E

   The correct sequence for DNA cloning steps is A, C, D, B, E.

   DNA cloning in recombinant DNA technology follows a logical order to create and verify recombinant molecules. This sequence ensures precise insertion of foreign DNA into a vector before host integration and validation.

   Correct Option Explained

   Option: A, C, D, B, E (Restriction digestion → Ligation → Transformation → Selection → Sequencing)

   This matches the standard protocol: First, restriction enzymes cut the insert DNA and plasmid to generate compatible ends (A). DNA ligase then joins them in vitro (C). The ligated product enters host cells via transformation (D). Transformants are selected using markers like antibiotic resistance (B). Finally, sequencing confirms the recombinant construct (E).

   Why Other Options Are Incorrect

   • B, C, D, A, E: Starts with selection, but no DNA exists to transform or select yet.

   • A, D, C, E, B: Places transformation before ligation; uncut DNA cannot form recombinants.

   • B, A, D, C, E: Begins with selection prematurely; digestion must precede any host involvement.

   Introduction to DNA Cloning

   DNA cloning steps in recombinant DNA technology enable scientists to amplify specific genes for research, medicine, and biotech applications. This process isolates, inserts, and verifies foreign DNA in host cells, forming the backbone of genetic engineering. Understanding the precise sequence is crucial for students preparing for competitive exams in molecular biology.

   Step-by-Step Process

   1. Restriction Digestion (A): Enzymes like EcoRI cut insert DNA and plasmid at specific sites, creating sticky or blunt ends for compatibility.

   2. In Vitro Ligation (C): T4 DNA ligase seals the insert into the vector, forming recombinant DNA.

   3. Transformation (D): Ligated DNA enters competent bacterial cells (e.g., E. coli) via heat shock or electroporation.

   4. Selection of Transformants (B): Markers (e.g., ampR gene) identify cells with recombinant plasmids; blue-white screening distinguishes recombinants.

   5. Confirmation by Sequencing (E): Sanger or NGS verifies insert orientation and integrity.​

   Step	Description	Purpose
   A	Restriction digestion of insert and plasmid	Generate compatible ends ​
   C	In vitro DNA ligation	Form recombinant plasmid ​
   D	Transformation of ligated DNA	Introduce into host ​
   B	Selection of recombinant host cells	Isolate successful clones ​
   E	Confirmation by sequencing	Validate construct ​

   This table summarizes the logical flow, preventing errors like non-recombinant vectors.

   Applications in Plant and Molecular Biology

   In plant sciences, DNA cloning aids gene function studies in photosynthesis or stress responses. For your exam prep in genetics and biotech, master this sequence to tackle matching questions on recombinant tools like PCR or electrophoresis.