PBL2: Restriction Mapping
1.
Define restriction endonuclease.
2.
Complete the following table
Site
Structure
pUC19
TaqI
T^CGA
4
"
DdeI
C^TNAG
6
"
PvuII CAG^CTG
2
"
HaeII RGCGC^Y
3
pBR322 BamHI G^GATCC
1
"
3.
# expected # expected
# of sites
(equal base (known base
observed
content)
content)
Plasmid
RsaI
GT^AC
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
a.
b.
c.
4.
Construct a restriction map of pBR322 showing the positions of the restriction sites for
EcoRI and these two enzymes.
Is this a unique map?
Suggest the identities of enzymes A and B.
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.
a. Construct a restriction map of pBR322 showing the positions of the restriction sites for
EcoRI and enzyme X.
b. Is this a unique map?
c. 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?