You cloned and sequenced a 5 Kbp gene obtained from the zebra fish genome. An an
ID: 201699 • Letter: Y
Question
You cloned and sequenced a 5 Kbp gene obtained from the zebra fish genome. An analysis with NEB cutter generated the following map:
The letters S, C, and K represent the restriction sites SpeI, ClaI, and KpnI, respectively. The numbers in parentheses represent the positions in Kbp from the origin (0).
To verify the computer generated map you perform a Southern hybridization on zebrafish genomic DNA digested with ClaI, KpnI or SpeI. The following table presents the sizes of the bands observed using two different probes (probes 1 & 2).
(a) These results indicate that the computer-generated map is missing one or more restriction sites. Indicate the restriction site (s) and it's (their) position (s) in parentheses.
(b) Which band size (s) observed on the Southern was (were) unexpected according to the computer generated map?
(c) To confirm the error, you wish to repeat the southern hybridization on a double digest of genomic DNA. Which enzymes would you use to confirm the error? Indicate the probe that would be used to confirm the presence of the missing site (s). Indicate the probe and the band size (s) which would would be expected if the site (s) is (are) truly present.
(d) A double digest of the genomic DNA with ClaI and SpeI is probed with probe 2. What band sizes are expected?
answer are 3a KpnI (1.3)
3b 2.1 Kb band in Kpn lane
3c Enzymes: K+C or K+S Probe: 1 Sizes: 1.0, 0.8 & 0.3 or 0.4 & 1.7
3d Band sizes: 1.5 and 3.5Kb
show me how to do it :)
S0.2) C(1.0) CI2.3)C2.6)S(30) K(3.4) C(4.5) Probe 1 Probe 2 (34) (4.8)Explanation / Answer
(a) From the results given, it is clear with the sizes of bands cleaved by ClaI is proper, while by Kpnl is mismatching with the NEB cutter data. If the process is double digestion as all the other restriction enzymes shows two or more restriction sites Kpnl shows only one. Hence, if the restriction site for Kpnl is in (1.3) position the band size width obtained is corrected.
(b) 2.1 Kb band in Kpn lane is unexpected as the NEB cutter data shows other sizes.
(c) As the error and unexpeccted size is obtained while using KpnI enzyme, I would like to prefer the same with other known restriction enzyme combination. For eg., Enzymes: K and C. If probe 1 is used and according to available restriction site data, the sizes of bands expected are 1.0, 0.8 and 0.3.
(d) The band size of 1.5 is obtained by cleavage by S(3.0) and C(4.5). Other one is 3.5 is obtained cleavage at C(1.0) and C(4.5)
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.