PBL2: Restriction Mapping

  1. Define restriction endonuclease.

  2. Complete the following table
    Plasmid Site Structure # of sites
    observed     		# expected
    (equal base
    content)         		# expected
    (known base
    content)
    pUC19 TaqI T^CGA 4

    " DdeI C^TNAG 6

    " PvuII CAG^CTG 2

    " HaeII RGCGC^Y 3

    pBR322 BamHI G^GATCC 1

    " RsaI GT^AC 3


  3. Plasmid pBR322 was first digested with EcoRI, which has a unique site within the plasmid. The plasmid was then treated with two different restriction enzymes (A & B), both as single and double digests. The sizes of the fragments obtained by each digest were determined by gel electrophoresis as follows:

    Enzyme A Enzyme B Both Enzymes
    650 bp 240 bp 240 bp
    1590 880 280
    2120 930 650

    		2310 880


    		1070


    		1240

    1. Construct a restriction map of pBR322 showing the positions of the restriction sites for EcoRI and these two enzymes.
    2. Is this a unique map?
    3. Suggest the identities of enzymes A and B.

  4. Plasmid pBR322 was first digested with EcoRI. The plasmid was then partially digested with restriction enzyme X. From the partial digest, fragments of the following sizes were found: 190, 350, 540, 680, 880, 1080, 1220, 1760, 2600, 3280, 3470, 3820 & 4360 base pairs.
    1. Construct a restriction map of pBR322 showing the positions of the restriction sites for EcoRI and enzyme X.
    2. Is this a unique map?
    3. Suggest the identity of enzyme X.

  5. What systematic process did you use to construct the restriction maps? Is this process an algorithm? If not, why not? If yes, is it of type NP?