Q15. In which functional domain is the patient’s APC sequence variant located?

Q14. Compare the molecular weight and relative abundance of the APC protein detected in the patient's colorectal tumour tissue and normal colon tissue. What do these results suggest about the effect of the sequence variant on the APC protein? (2-3 sentences) (3 marks)

Q13. Do some research. What is the expected molecular weight of the full-length human APC protein?

The reaction resulted in successful amplification of exon 7 of the APC gene, producing a single PCR product of the expected amplicon size. The PCR product was excised from the agarose gel, purified, and subjected to Sanger DNA sequencing for further analysis. Unfortunately, a laboratory technician made an error when setting up the Sanger sequencing reaction. Consequently, the sequencing reaction terminated at the first guanine (G) position, and no sequence data was obtained beyond the eighth nucleotide. The erroneous sequencing results are shown in the chromatogram in Figure 2.

Figure 2. Sanger sequencing chromatogram generated for the APC PCR amplicon.

The technician successfully repeated the Sanger sequencing

experiment.

Use the Clustal Omega

sequence alignment tool to perform a pairwise alignment between:

1. the DNA sequencing results for the patient's colorectal tumour tissue, and

2. the

   NCBI RefSeq sequence for

   Homo sapiens APC exon 7 (NG_008481.4).

Q8.

Upload a screenshot of the pairwise alignment output from

Clustal Omega to the file upload box below. (2 marks)

Q10. How would you classify the patient’s sequence variant at the DNA level?

Q11. How would you classify the patient’s sequence variant at the protein-coding level?

Q12. Concisely describe the patient’s sequence variant using Human Genome Variation Society (HGVS) nomenclature, including both the DNA-level and protein-level notation, in a manner suitable for a scientific report. (4 marks)

Q6. Select a suitable extension time for the PCR reaction.

Q4. Use the primer nucleotide sequences and NCBI RefSeq for the APC gene from Homo sapiens (NG_008481.4) to determine the expected length of the PCR product. The RefSeq is available in the assessment section on Moodle and can be accessed via this direct link.