Question
List the steps of recombinant DNA technology in the correct sequence. Give one example of a vector used at each relevant step.
This appeared in NEET 2023 and has consistent weightage in CBSE Class 12 board exams. The question tests whether you know the sequence — not just the names of the steps.
Solution — Step by Step
We start by extracting DNA from the source organism — this could be human insulin gene DNA or Bt toxin gene from Bacillus thuringiensis.
The DNA is released by lysing cells using lysozyme (bacteria), cellulase (plants), or chitinase (fungi). RNA and proteins are removed using RNase and protease respectively, leaving purified DNA.
Restriction enzymes are the molecular scissors. The same enzyme cuts both the donor DNA and the vector so that compatible sticky ends are produced.
For example, EcoRI cuts at 5'...G↓AATTC...3' — leaving a 4-nucleotide overhang. The vector here is typically a plasmid like pBR322, which carries two restriction sites (EcoRI and HindIII) and two antibiotic resistance genes (amp^R and tet^R).
If the isolated gene quantity is very low, we amplify it using Polymerase Chain Reaction before ligation.
PCR uses Taq polymerase (thermostable), specific primers, and cycles of denaturation → annealing → extension. A single copy becomes billions in 30–40 cycles.
The cut gene and cut vector are joined by DNA ligase — the molecular stapler. It forms phosphodiester bonds between the sticky ends.
The result is a recombinant DNA molecule (rDNA). Common vectors: plasmids (pBR322), bacteriophage lambda (λ), cosmids for large DNA inserts, and Ti plasmid from Agrobacterium tumefaciens for plant gene transfer.
The recombinant plasmid is introduced into a suitable host — usually E. coli.
Bacteria are made competent (able to take up DNA) by treating with calcium chloride (CaCl₂) solution on ice, then heat-shocked at 42°C. This creates transient pores in the membrane. For plant cells, the Ti plasmid vector is used via Agrobacterium-mediated transformation.
Not every bacterium takes up the recombinant plasmid. We need to identify which colonies carry the insert.
Using insertional inactivation with pBR322: if the gene is inserted into tet^R, the recombinant colonies lose tetracycline resistance but remain ampicillin resistant. Colonies that grow on ampicillin plates but NOT on tetracycline plates carry the recombinant DNA. Those are our transformants.
Why This Works
The entire logic of rDNA technology rests on restriction enzymes being sequence-specific. Because both vector and insert are cut by the same enzyme, they produce complementary sticky ends — they’re designed to join together.
The host organism (E. coli is preferred because it divides every 20 minutes) replicates both its own DNA and the recombinant plasmid. So every daughter cell carries the foreign gene — giving us large quantities of the protein product.
Selection is the most commonly tested step because it’s non-obvious. You’re essentially using antibiotic resistance as a reporter — bacteria tell you whether they’ve taken up the recombinant DNA by dying (or not dying) on different antibiotic plates.
Alternative Method — Selectable Marker: Blue-White Screening
Instead of pBR322, many modern vectors use pUC19 with a lacZ gene (codes for β-galactosidase).
If the foreign gene inserts into lacZ, it disrupts the gene. On X-gal plates:
- White colonies = recombinant (gene inserted, lacZ non-functional, no blue colour)
- Blue colonies = non-recombinant (lacZ intact, cleaves X-gal → blue product)
In NEET MCQs, “blue-white screening” always means the white colonies are the ones you want — the recombinants. This trips up many students who assume “selected = blue.”
Common Mistake
Students confuse transformation (inserting DNA into bacteria) with transfection (inserting DNA into eukaryotic cells). NEET questions specifically test this distinction. Also: many write that CaCl₂ treatment alone transfers DNA — it doesn’t. CaCl₂ makes cells competent, but the actual transfer happens during the heat shock step. Both steps are needed.
Isolation → Cutting (RE) → Amplification (PCR) → Ligation → Transformation → Selection
Vector examples: pBR322 and pUC19 (bacteria), Ti plasmid (plants), λ phage (large inserts)
The complete sequence in one line: we isolate the gene, cut it and the vector with the same restriction enzyme, join them with ligase, push the recombinant into a host, then screen using antibiotic resistance or blue-white selection.