A Sickle Cell Anemia Detection Simulation: PCR, Restriction Enzyme Digest, and Electrophoresis In this activity, the genotyping of a newborn baby will be simulated with respect to the sickle cell mutation. Does the baby have a wild-type beta-globin gene, a mutation on one allele (carrier), or a mutation on both alleles (homozygous)? Known samples of wild-type and homozygous mutant beta globin will be used for comparison. 1. Write down the known partial DNA sequence for the wild-type beta-globin gene as seen below. This sequence represents the portion of the beta-globin gene that has been amplified using PCR. Typically, amplified sequences will be longer than this example. and some restriction enzymes need longer sequences to function. However, for ease in this activity, the sequences are short. Wild-type: 5' CTG ACT CCT GAG 3: 3' GAC TGA GGA CTC 5' 2. Beside the wild-type sequence, write the DNA sequence for the known mutation associated with sickle cell anemia. Tanio 2 Homorygous mutant: 5' CTG ACT CCT GTG
3 ′3' GAC TGA GGA CAC 5' 3. Determine the recognition site and cleavage pattern of Dde 1 by referring to the restriction enzyme table provided in the background section (see Table 1). Remember that the