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From Sequence to Protein: The IDH Story
Isocitrate dehydrogenase, IDH, is a protein that catalyzes a reaction in the citric acid cycle, giving the cell both NADPH and 2-oxoglutarate, two molecules needed for the synthesis of specific lipids and amino acids (Purves et al., 1998). In this research project, we will isolate, clone, and express the five genes that encode the IDH protein in the yeast Saccharomyces cerevisiae. The entire yeast genome has been sequenced and catalogued. Below are links to the accession numbers, DNA nucleotide coding sequences, and the amino acid sequences.
Furthermore, each gene makes an enzyme that associates with one of two coenzymes: NADH or NADP. IDH1 and IDH2 associate with NADH, while IDP1, IDP2, and IDP3 associate with NADP (Cherry et al., 2000).
As explained in the lab schedule, we purified genomic DNA from the yeast and then PCR amplified each of the genes using primers developed from the known DNA sequences of the particular genes. One primer will be the same as the first 5' 20 bases of the gene; these bases will be complementary to the 3' bases of one DNA strand. The second primer must be made complementary to the 3' end of the strand that is known. When designing the primers, their length and optimal melting temperature were considered. Melting temperatures should be around 60 degrees Celsius and primer lengths should be 18-20 base pairs in order for them to be specific enough for the portion of DNA we want to amplify (Campbell, 2002). Our primer sequences can be found here.
The next step was to clone each gene into the vector in the correct orientation.
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