Biology · Biotechnology: Principles and Processes · NEET
pBR322 is the standard E. coli cloning vector shown in NCERT Figure 9.4. It carries four key parts: ori (starts replication and controls copy number), rop (protein for plasmid replication), and two antibiotic-resistance selectable markers, ampR (ampicillin) and tetR (tetracycline). Memory hook: "AMP and TET are the two guards, ORI and ROP run the copy machine." NEET has asked pBR322 almost every year, so knowing each labelled part is worth easy marks.
pBR322 map based on NCERT Figure 9.4: ori and rop run replication, while ampR and tetR are the two antibiotic-resistance selectable markers. The BamHI site sits inside tetR and the PstI site inside ampR, so inserting DNA at either site inactivates that resistance.
Your doubts, answered
What do ampR and tetR actually mean on pBR322?
ampR is the gene giving resistance to the antibiotic ampicillin, and tetR is the gene giving resistance to tetracycline. Both are selectable markers. They let you find bacteria that took up the plasmid, because only those cells can survive on a plate that contains the antibiotic. NCERT Figure 9.4 labels both genes on the pBR322 circle.
What is the difference between ori and rop in pBR322?
ori (origin of replication) is the sequence where DNA replication begins, and it also controls the copy number (how many copies of the plasmid are made inside the cell). rop codes for the proteins involved in the replication of the plasmid. In the NEET 2024 diagram question, X = ori (copy number) and Y = rop (replication protein). Do not confuse either with antibiotic resistance, which comes only from ampR and tetR.
Which gene does BamHI cut, and what happens?
In pBR322 the BamHI restriction site lies inside the tetracycline resistance gene (tetR). If you ligate foreign DNA there, that gene is disrupted. This is called insertional inactivation, so the recombinant plasmid loses tetracycline resistance but keeps ampicillin resistance. That difference is exactly how you tell recombinants from non-recombinants.
Which gene does PstI cut?
The PstI site lies within the ampR (ampicillin resistance) gene. So if a gene is inserted at the PstI site, the ampicillin resistance is inactivated instead of tetracycline. NEET 2021 tested exactly this: inserting a gene at the PstI site inside ampR means the plasmid can no longer confer ampicillin resistance to the host.
Is pBR322 used in animals or in bacteria?
pBR322 is a bacterial (E. coli) cloning vector only. For animal cells such as human lymphocytes, disarmed retroviruses are used; for plant cells, the Ti plasmid of Agrobacterium is used. NEET 2018 gave all four options together to test this exact confusion.
What does the name pBR322 stand for?
For NEET you only need the function, but for interest: p = plasmid, BR = Bolivar and Rodriguez (the scientists who built it), and 322 is a lab identification number. NCERT does not ask you to recall the name meaning, only the labelled parts ori, rop, ampR and tetR.
⚠️ The NEET trap ✗ ori and rop give antibiotic resistance in pBR322. ✓ Antibiotic resistance comes only from ampR and tetR. ori controls the copy number and start of replication; rop codes for proteins involved in plasmid replication. 🧠 In the NEET 2024 diagram question, the trap options attach 'antibiotic resistance' to X or Y. Resistance is never ori or rop. ori = copy number, rop = replication protein.
Real NEET questions
NEET 2019 (Odisha)
The two antibiotic resistance genes on vector pBR322 are for
A · Ampicillin and Tetracycline ✓
B · Ampicillin and Chloramphenicol
C · Chloramphenicol and Tetracycline
D · Tetracycline and Kanamycin
Solution: pBR322 carries two selectable-marker resistance genes, ampR (ampicillin) and tetR (tetracycline), as named in NCERT Figure 9.4. Chloramphenicol and kanamycin are only general examples of markers, not the genes on pBR322.
NEET 2026 (Phase 1)
Insertion of a foreign DNA at BamHI site in an E. coli cloning vector pBR322 results in the loss of antibiotic resistance towards:
A · Ampicillin and tetracycline
B · Ampicillin
C · Tetracycline ✓
D · Gentamycin
Solution: The BamHI site lies within the tetracycline resistance gene. Inserting foreign DNA there causes insertional inactivation, so the recombinant plasmid loses tetracycline resistance but keeps ampicillin resistance for selection.
NEET 2024
The following diagram shows restriction sites in E. coli cloning vector pBR322. Find the role of 'X' and 'Y' genes:
A · X controls the copy number of the linked DNA and Y codes for protein involved in replication of the plasmid ✓
B · X is for protein involved in replication of plasmid and Y for resistance to antibiotics
C · X is responsible for recognition sites and Y for antibiotic resistance
D · X is responsible for resistance to antibiotics and Y for protein involved in replication of the plasmid
Solution: X = ori, which controls the copy number and starts replication; Y = rop, which codes for proteins involved in plasmid replication. Antibiotic resistance is a separate function of ampR and tetR, so options linking X or Y to resistance are wrong.
Solved Biotechnology: Principles and Processes NEET PYQs
Try the real previous-year questions from this chapter — each with the answer and a full solution.
How many antibiotic resistance genes does pBR322 have?
Two: ampR for ampicillin and tetR for tetracycline. Both act as selectable markers to identify bacteria that took up the plasmid.
Which sequence controls the copy number in pBR322?
The origin of replication (ori). It is where replication starts and it also controls how many copies of the linked DNA are made. NEET 2020 tested this directly.
Does rop give antibiotic resistance?
No. rop codes for proteins involved in the replication of the plasmid. Resistance comes only from ampR and tetR.
Why is BamHI used with pBR322 in NCERT?
Because the BamHI site is inside the tetracycline resistance gene, so inserting DNA there inactivates tetR. This gives a simple way to select recombinants: they grow on ampicillin but not on tetracycline.
Can pBR322 be used to put a gene into human cells?
No. pBR322 works only in bacteria (E. coli). Human/animal cells use disarmed retroviruses, and plant cells use the Ti plasmid.
