Figure 6. Western blot detection of Bak, Bax and Bcl-2 proteins in patient-derived colorectal cancer intestinal epithelial cells (IECs) and normal IECs.

Apoptosis was then assessed functionally following camptothecin treatment by examining DNA fragmentation using agarose gel electrophoresis and executioner caspase activation assays. The executioner caspase activation assay demonstrated robust executioner caspase activation in the normal IECs following treatment, whereas no executioner caspase activation was detected in the patient-derived colorectal cancer IECs.

Figure 7. Three potential agarose gel electrophoresis results (A-C) for the DNA fragmentation assay. M = DNA ladder, 1 = normal IECs without camptothecin treatment, 2 = normal IECs following camptothecin treatment, 3 = colorectal cancer IECs following camptothecin treatment.

The colorectal tumour tissue and normal colon tissues resected from the patient were dissociated into single cell suspensions. Intestinal epithelial cells (IECs) were isolated from the suspensions and cultured in vitro. The IECs were labelled with a GFP-tagged anti-β-catenin antibody and DAPI. The results are shown in Figure 5.

Figure 5. Fluorescent microscopy images showing β-catenin localisation in intestinal epithelial cells (IECs) isolated from patient-derived colorectal tumour tissue and normal colon tissue.

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 thymine (T) 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.

Following surgical resection, samples of the tumour tissue and adjacent normal colon tissue were fixed in formalin and sectioned into thin slices for histological analysis. The tissue sections were stained with haematoxylin and eosin (H&E) and examined by a pathologist using light microscopy to assess tissue architecture and cellular morphology. Representative histological images from the patient’s normal colon tissue and tumour tissue are shown in Figure 1.

Figure 1. Histological analysis of patient-derived normal colon tissue (A) and colorectal tumour tissue (B) stained with haematoxylin and eosin (H&E).

Following surgical resection, samples of the tumour tissue and adjacent normal colon tissue were fixed in formalin and sectioned into thin slices for histological analysis. The tissue sections were stained with haematoxylin and eosin (H&E) and examined by a pathologist using light microscopy to assess tissue architecture and cellular morphology. Representative histological images from the patient’s normal colon tissue and tumour tissue are shown in Figure 1.

Figure 1. Histological analysis of patient-derived normal colon tissue (A) and colorectal tumour tissue (B) stained with haematoxylin and eosin (H&E).

Following ligation, the researcher transformed a competent strain of

Q27. The researcher plans to set up a 20 µL ligation reaction with a total plasmid mass of 50 ng. What is the mass of the digested insert DNA that the researcher must add to the ligation reaction to achieve a 3:1 ratio of insert to plasmid? Record your answer to two decimal places and show your calculations.

Q25. From the available restriction enzymes, select two appropriate restriction enzymes for cloning the exon 7 insert into your selected plasmid. 

Q24. From the available expression plasmids, select an appropriate plasmid for cloning the exon 7 insert.