• A. Visualisation of amplified product
• B. PCR machine run
• C. DNA isolation
• D. Loading of PCR run
• E. PCR reaction setup in tube

Choose the correct answer from the options given below:

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

   Correct Answer: Option 2 (C, E, D, B, A)

   PCR follows a logical lab workflow: first isolate DNA, then set up the reaction, load it into the machine, run amplification cycles, and finally visualize results. This sequence ensures contamination-free amplification and verification.

   Step-by-Step PCR Sequence

   Standard PCR protocol matches these steps precisely.

   • C. DNA isolation: Extract pure DNA template from sample to avoid inhibitors.​

   • E. PCR reaction setup in tube: Mix template, primers, dNTPs, Taq polymerase, buffer in a tube.​

   • D. Loading of PCR run: Transfer tube to thermal cycler (PCR machine) block.​

   • B. PCR machine run: Execute cycles (denaturation ~95°C, annealing ~55°C, extension 72°C; 25-35 repeats).​

   • A. Visualisation of amplified product: Run gel electrophoresis, stain with ethidium bromide, image under UV.​

   Option Analysis

   Option	Sequence	Issue	Correct?
   1	A, B, C, D, E	Starts with visualization (last step) before any setup or run ​	No
   2	C, E, D, B, A	Matches exact lab order: isolate → setup → load → run → visualize	Yes
   3	B, A, D, C, E	Runs machine before DNA isolation or setup; visualizes too early	No
   4	E, B, D, C, A	Runs machine (B) right after setup, skipping loading (D); isolates DNA last	No

   Only option 2 follows chronological lab protocol.

   ---

   Introduction to PCR Steps Sequence

   The PCR steps sequence in molecular biology follows a precise order: DNA isolation, PCR reaction setup, loading of PCR run, PCR machine run, and visualisation of amplified product. This NEET question tests understanding of the complete PCR protocol workflow.​

   Detailed PCR Protocol Breakdown

   PCR amplifies specific DNA segments exponentially through thermal cycling.

   1. DNA Isolation (C): Purify genomic/sample DNA using kits or phenol-chloroform.​

   2. PCR Reaction Setup (E): In ice-chilled tubes, add 1-500 ng template, primers (0.1-0.5 μM each), dNTPs, Taq polymerase.​

   3. Loading PCR Run (D): Place tubes in thermal cycler wells; program cycles.​

   4. PCR Machine Run (B): 94°C denaturation, 55°C annealing, 72°C extension (25-35 cycles).​

   5. Visualization (A): Agarose gel with EtBr; UV transilluminator confirms band size.​

   Common PCR Errors to Avoid

   • Skipping isolation leads to inhibitors.

   • Setup on bench (not ice) causes non-specific amplification.

   • Wrong sequence (like options 1,3,4) fails in real labs.​

   NEET Exam Tips

   Memorize C-E-D-B-A as “Clean DNA, Easy setup, Drop in machine, Blast cycles, Analyze.” Practice with cycle parameters for scoring.