59. You restriction map a vector using Bam HI, Eco RI and Hae III and obtained
fragments of the following sizes. Draw a restriction map of the vector and
determine which of the following statements is correct.
Results:
Bam HI alone: 4,000bp;
Eco RI alone: 4,000bp;
Hae III alone: 3,000bp and 1,000bp;
Bam HI and Eco RI: 3,500bp, 500bp;
Bam HI and Hae III: 2,300bp, 1,000bp, 700bp;
Eco RI and Hae III: 2,8000bp, 1,000bp, 200bp;
Bam HI, Eco RI, and Hae III: 2,300bp, 1,000bp, 500bp, 200bp
a. There are three Hae III sites in this vector
b. Bam HI is 1,000bp away from Eco RI
c. Bam HI is present within the smaller fragment from the Hae III digestion
d. Eco RI is present within the larger fragment from the Hae III digestion
The vector is circular, 4000 bp total, with one BamHI site, one EcoRI site (500 bp apart), and two HaeIII sites. HaeIII cuts produce a 3000 bp larger fragment and 1000 bp smaller fragment alone. BamHI + EcoRI yields 3500 bp and 500 bp, confirming the enzymes are 500 bp apart. The correct statement is d. Eco RI is present within the larger fragment from the Hae III digestion.
Map Construction
Single digests confirm: BamHI (1 site), EcoRI (1 site), HaeIII (2 sites). Double digest BamHI + EcoRI (3500 + 500 bp) places sites 500 bp apart on the circle. BamHI + HaeIII (2300 + 1000 + 700 bp) and EcoRI + HaeIII (2800 + 1000 + 200 bp) show HaeIII fragments subdivided differently by each enzyme. Triple digest (2300 + 1000 + 500 + 200 bp) reveals four pieces, with the intact 1000 bp HaeIII fragment and the 3000 bp split into 2300 + 500 + 200 bp. Positions: BamHI at 0 bp, EcoRI at 500 bp, HaeIII sites at ~700 bp and ~2700 bp (within 0-3000 bp arc).
Option Analysis
-
a. Three Hae III sites: Incorrect. Single HaeIII gives two fragments (3000 + 1000 bp), indicating exactly two sites on circle. Triple digest shows four fragments total, not five from three sites.
-
b. Bam HI 1,000bp from Eco RI: Wrong. BamHI + EcoRI produces 500 bp fragment, so distance is 500 bp (shorter arc), not 1000 bp.
-
c. Bam HI in smaller Hae III fragment: False. BamHI + HaeIII yields 2300 + 700 bp (from larger 3000 bp HaeIII) + intact 1000 bp, placing BamHI in larger fragment.
-
d. Eco RI in larger Hae III fragment: Correct. EcoRI + HaeIII gives 2800 + 200 bp (from 3000 bp) + intact 1000 bp; 500 bp from triple confirms EcoRI subdivides larger fragment.
This CSIR NET question tests double/triple digest logic for circular plasmids, emphasizing fragment additivity and site localization within single-enzyme pieces.
