Q.82 If the plasmid given below is digested with restriction enzymes HindIII and
EcoRI, considering complete digestion, how many DNA fragments will be
released?

Plasmid digestion with HindIII and EcoRI releases 3 DNA fragments upon complete digestion.

Understanding Restriction Digestion

Circular plasmids like pMC produce distinct fragment numbers based on cut sites. HindIII cuts at two positions, linearizing the plasmid into one large piece initially. EcoRI cuts at one position, but combined action at all three non-overlapping sites divides the circle into three separate linear fragments.

Analyzing the Diagram

The diagram shows a circular pMC plasmid with HindIII sites opposite each other and one EcoRI site offset between them. Complete digestion means every recognition sequence cleaves fully, yielding fragments corresponding to arcs between cuts: one between the two HindIII sites, one from HindIII to EcoRI, and one from EcoRI to the other HindIII.

Step-by-Step Fragment Count

• HindIII alone: 2 sites yield 2 fragments (circular DNA rule: cuts = fragments).

• EcoRI alone: 1 site yields 1 fragment (linearized plasmid).

• HindIII + EcoRI: 3 total sites yield 3 fragments, as no sites coincide.

Plasmid digested with HindIII and EcoRI is a key question in molecular biology for CSIR NET Life Sciences preparation. This complete digestion scenario tests understanding of restriction enzyme mapping on circular DNA.

Key Concept: Restriction Enzymes on Plasmids

Restriction enzymes like HindIII (cuts A|AGCTT) and EcoRI (G|AATTC) recognize palindromic sequences. For circular plasmids, complete digestion means all sites cleave, and fragment number equals cut sites if non-overlapping. The pMC plasmid diagram reveals two HindIII sites and one EcoRI site, totaling three cuts.

Detailed Solution Breakdown

Visualize the plasmid circle:

• HindIII at positions ~0° and ~180°.

• EcoRI midway between one HindIII pair.

Cuts divide the circle into:

• Fragment 1: HindIII to HindIII (large arc).

• Fragment 2: HindIII to EcoRI.

• Fragment 3: EcoRI to HindIII.

Thus, 3 DNA fragments release, separable by agarose gel electrophoresis.

Options Explanation (Typical MCQ Format):

• Option 1 (1 fragment): Incorrect—single cut linearizes (EcoRI alone); multiple cuts fragment further.

• Option 2 (2 fragments): Incorrect—matches HindIII alone; ignores EcoRI.

• Option 3 (3 fragments): Correct—total unique sites.

• Option 4 (4 fragments): Incorrect—assumes overlap or extra site, not shown.

CSIR NET Exam Tips

Practice restriction mapping: Single digest gives site count; double digest compares patterns. Tools like gel electrophoresis confirm sizes summing to plasmid length. For plasmid digested with HindIII and EcoRI, always count visible sites on diagrams.