Determining the size of gene fragments
1) Alul restriction enzyme cuts the protease gene at restriction sites with base position 62 and position 1149 respectively. Considering the fact that the gene has 1216 base pairs, three possible fragments are generated with Alul restriction digestion.
The first digestion fragment length ranges from base pair 1 to base pair 62= 61 base pairs.
The second fragment length ranges from base pair 62 to base pair 1149 in length=1087 base pairs
The third fragment ranges from base pair 1216 to base pair 1149 in length= 67 base pairs.
2) Sa/l restriction enzyme cuts the protease gene at base pair 355 and 473 respectively. Three fragments are generated from this restriction digestion.
The first fragment length ranges from base pair 1 to base pair 355 in length = 354base pairs.
The second Fragment length ranges from base pair 355 to base pair 973 in length =618 base pairs.
The third fragment length ranges from base pair 973 to base pair 1216 in length= 243base pairs
3 )Kpnl restriction enzyme cuts the protease gene at base position 231 and 473 respectively.
Three fragments are generated from this restriction digestion.
The first fragment is from base pair 1 to base pair 231 in length =230base pairs.
The second fragment length ranges from base pair 231 to 473= 242base pairs .
The third gene fragment length ranges from base pair 1216 to 473 in length that equals 743 bases.
Pstl restriction enzyme cuts the protease gene fragment at restriction site at position 1128. Two fragments are generated by this restriction enzyme digestion. The first fragment ranges in length from base pair 1 to base pair number 1128 = 1127 base pairs
The second fragment ranges in length from base pair number 1128 to 1216=88 base pairs.
Which among the fragments of restriction digestion will you try to clone?
Among the restriction fragments digests, the most ideal for use in cloning are the fragments with overhanging ends or sticky ends. These fragments are the fragments generated by the restriction enzymes Sa/l, Kpnl and pstl. All the three restriction enzymes, Sal/l, Kpnl and Pstl generate fragments with sticky end (Siegmund & Yakir, 2007).
Sticky ends are easier to clone/ join together with other DNA molecules with DNA ligase enzyme as compared to blunt ends .Since sticky ends are complementary, they allow the joining of DNA molecule in only one direction, which is highly desirable in cloning exercises. The yield of positive mutants in cloning is also higher with the use of sticky ends than with blunt ends (Siegmund& Yakir, 2007). This means that only fragments obtained from restriction digestion by sa/l kpnl and pstl are the only ones most ideal for cloning.
Determine which gene which will be detected by the four probes in the experiment
Probe L with base pair 4.7 to 5 will detect regA gene because this gene is (complementary) found within the same range of base pairs from the E coli restriction enzyme site as the probe.
Probe M will detect gene gep A because the gene sequence complementary with the probe meaning the probe will hybridize with it.
Probe N will hydride/ detect gep C gene because they are within the same positions in the genome (complementary) to the probe sequence.
Probe O will detect gene gep C because of complementary sequence since they are within the same position in the genome map.
Calculate the size of fragments generated by the four probes in the experiment
Probe L will detect part of gene reg A . The size of the gene fragments detected by probe m considering its relation in the map is 4.7 to 5 which are 0.3 kilo base pairs or 300 base pairs.
The length of fragment detected by probe m
Probe M 5.9-6.2= 0.3 kilo base pairs = 300 base pairs.
Probe N will detect a 300 base pair fragment of geb B gene
Probe O will detect a gene fragment of gep
C with length 7.4-7.8 equal 0.4 kilo base pairs or 400 base pairs.
Siegmund D & Yakir Benjamin (2007) the statistic of gene mapping London: Springer.