1. DNA-based cases

Develop a case study, research problem, etc. that can be addressed using restriction enzyme digestion, PCR, gel electrophoresis, Southern blotting, dot blotting, sequence alignment, or phylogenetic trees. The general steps involved include:Find the relevant DNA sequence(s). This can be done by searching the GenBank database using key words. For example, the human hemoglobin gene for the sickle cell anemia case study was obtained by using the key words “hemoglobin” and “sickle”. This search actually returned dozens of sequence files that had been submitted to GenBank with notations containing the key words; one of these files was the complete human hemoglobin gene.

a. Determine how the sequence should be modified, if at all, to fit the case. Do you need to use only a portion of the gene? Do you need to create a wild type and/or a mutated version? This is often the most difficult part of preparing the case and requires some prior knowledge about the system and/or a literature review. Often the GenBank files will include information about the location of key mutations. Save sequence files generated as text-only files.

b. If restriction enzymes are needed, generate the enzyme site files (again, saved as text-only files).

c. For PCR, generate primer files. Determine from the literature which region of the DNA to amplify and which forward and reverse primers sequences will be used Create a text file containing both primer sequences (first the forward, then the reverse sequence) separated by a carriage return. Primers should be written in the conventional format, i.e. 5’ to 3’; the reverse primer sequence will be complementary to that region of the target DNA sequence. For quantitative PCR in 96-well format, a viral load value can be added to the end of the sequence; this value will be reported if the Viral Load option is selected for 96-well PCR data.

d. For Southern blotting or dot blotting, determine what portion of the sequence to use as a probe, e.g. near polymorphisms affecting restriction enzyme sites (for Southern blotting) or spanning the region containing the mutation (for dot blotting) Again, this requires literature references. Generate the probe file by copying from the sequence file and pasting into a new file or by typing a new text file. Save the probe as a text-only file.

e. Filter all of the sequence files using the GenBank filter in the program and save the filtered files.

IMPORTANT NOTE: DNA sequence files should only contain the letters A, C, G, and T. Any other letters (other than N, indicating an unknown base) should be removed to avoid an error message.

2. Protein-based cases

The steps involved for generating protein cases are very similar to those for DNA-based cases. However, unlike probes, primers and restriction enzyme where the precise sequence used in a real lab setting can be obtained, proteins are usually detected by antibodies. Antibodies may bind conformational determinants, dependent on the shape of the protein, which the Case It! software cannot simulate. Therefore, antibody files are a short amino acid sequence from one or more proteins. It is sometimes possible to find antibody binding sites reported in the literature, but in some cases it is necessary to just use a short peptide unique to that protein. The contents of the antibody file do not appear in the upper left corner of the data screen.

The Case It! simulation will calculate the molecular weight in kD based on the amino acid sequence of the protein, and the proteins will migrate on the gel according to this calculated size. If proteins samples are glycoproteins, and carbohydrates contribute to the apparent size, the weight value of the carbohydrate must be added to the calculated size in order for the protein to run accurately on the gel. Indicate this value at the end of the protein sequence between + signs. There are additional codes that are placed in the protein and antibodies files to prescribe the strength of the reaction, in order to achieve some variability among results. Contact mark.s.bergland@uwrf.edu for more information if you are developing these types of cases.