Question

QUESTION 6 Assume you are studying a protein-coding gene, ACEX, which includes 4 exons as illustrated in the gene map below. The 5' UTR and 3' UTR segments are each 25 bp long. Exons 1 thru 4 are 100, 200, 300, 400 bp long, respectively. Each intron is 200 bp each. The locations of the relevant EcoRI sites within the ACEX locus are indicated, but the location of other restriction enzyme sites (like BamHI) are not shown." EcoRI probe EcoRI 25 100 400 25 200 You carried out a Southern Blot analysis using samples from different types of healthy cells from the same individual: pancreatic cells and brain cells. Based on the information above, which of the Southern Blots below (I-V), shows the most likely results?

Answer 1

Q6)

Blot IV

The ACEX locus has two EcoRI sites, one at the beginning of Exon 2 and another at the end of 3' UTR. These two sites are 1325 bp apart on the DNA molecule, as Exon 2 is 200 bp, Intron 2 is 200 bp, Exon 3 is 300 bp, Intron 3 is 200 bp, Exon 4 is 400 bp and the 3' UTR is 25 bp long (200 + 200 + 300 + 200 + 400 + 25 = 1325).

The probe is complementary to Exon 4 and will bind any DNA molecule that includes that sequence.

Since we know that EcoRI treatment produces a 1325 bp fragment (in both Pancreatic and Brain DNA as the genome is the same in both these tissues.); Blot II and Blod IV are possibly those that are correct.

However, a DNA fragment that is bound by the probe against Exon 4 of ACEX cannot be longer than 1325 bp, as EcoRI will cleave all DNA molecules at those two sites. Therefore, the correct Blot cannot be Blot II as the EcoRI + BamHI digested bands are shown to be 1700 bp long.

Blot IV is the correct answer.

Q7)

• Based on this information, the exons of the gene shown above include a total of 1050 nucleotides.

Since Exon 1 is 150 bp, Exon 2 is 200 bp, Exon 3 is 300 bp, Exon 4 is 400 bp, total number of nucleotides in the Exons is 150 + 200 + 300 + 400 = 1050

• The mRNA will include 350 (= 1050/3) codons.
• The number of amino acids in the protein translated from the mRNA is 349.

Since the last codon is the Stop Codon, it does not code for an Amino Acid. Therefore, 350 - 1 = 349 codons code for amino acids.

• The approximate mass of the full-sized, non-processed protein will be 38.39 kDaltons

Since there are 349 amino acids, each with a mas of 110 Da, the total mass of the non-processed protein = 349 * 110 Da = 38390 Daton = 38.39 kDaltons

• The first amino acid at the N terminus will be Methionine.

Methionine (Formyl-Methionine in Prokaryotes) is usually the N terminus Amino acid. In mature proteins, it is sometimes cleaved as part of the signal sequence/targeting sequence.

Q8)

Please include an image of the Northern Blot for an answer.

Q11)

Which probe or probes would be able to detect a 1000 bp fragment in a Southern Blot? (Eco RI digested DNA from Brain)

8. Probe A only

Since a DNA fragment 1000 bp is assayed, it can only be the fragment between the first and second EcoRI sites. Only Probe A is complementary to this region.

Which probe or probes would be able to detect a 1000 bp fragment in a Southern Blot? (EcoRI digested DNA from Liver)

8. Probe A only

Since DNA is the same in all cells, in this scenario as well, only Probe A will be able to detect the 1kb fragment.

Which probe or probes would be able to detect a 650 bp fragment in a Southern Blot? (Eco RI digested DNA from Brain)

1. Probe B and Probe C.

Both Probe B and Probe C are complementary to the 650 bp fragment produced by EcoRI digestion (the region between EcoRI site 2 and site 3)

Which single probe would be able to detect a 650 bp fragment and a 1000 bp fragment simultaneously in a Southern Blot

6. None; neither one of the probes (A, B, or C) would work.

This is because none of the probes is complementary to both the 1000 bp and the 650 bp fragments. Probe A is complementary only to the 1000 bp fragment while Probes B and C are complementary to the 650 bp fragment only.

Which probe or probes should be able to detect a fragment in a Northern Blot.
(RNA isolate from Brain tissue)

7. Probe B only

Since Northern Blots assay for RNA and only Probe B is complementary to an Exonic sequence, Probe B is the only Probe that will bind to the Nucleic acid in a Northern Blot.

Which probe or probes should be able to detect a band on a Western Blot

6. None; neither one of the probes (A, B, or C) would work.

Since these are DNA probes, they would not be able to bind the resultant Protein molecules that are assayed in a Western Blot.

Which probe or probes should be able to detect a fragment in a Northern Blot.
(RNA isolate from Liver tissue)

6. None; neither one of the probes (A, B, or C) would work.

Since this gene is not expressed in Liver cells, no RNA will be produced from the Liver corresponding to this gene. Therefore, none of the probes will be able to detect a fragment on a Northern blot of Liver tissue extract.

Which probe or probes would be useful in a Southern Blot analysis of the ACME locus but would NOT be useful in a Northern Blot Analysis.

3. Probe C only.

Since Probe C lies outside of the region of DNA that is transcribed, it ill be useful in Southern Blotting, where DNA is assayed, but not in Northern Blotting where RNA is used and therefore, Probe C has no complementary sequence.