(a) The SNP mutation responsible for sickle-cell disease changes amino acid 6 in hemoglobin β from glutamic acid (E) to valine (V). This polymorphism is PCR-amplified by primers complementary to flanking sequences that do not vary between alleles. (b) Sequencing of PCR products. The sequence of the PCR product from a heterozygous carrier shows both the normal (HbβA) and mutant (HbβS) nucleotides at the position of the substitution (black arrow).
Part A shows PCR-amplifying alleles of the Hb beta gene. The Hb beta superscript A shows a double-stranded sequence. The sequence in the strand from 5 prime to 3 prime reads, ACT CCT GAG (A highlighted) GAG AAG. The sequence in the strand from 3 prime to 5 prime reads, TGA GGA CTC (T highlighted) CTC TTC. The above sequence reads, 4, 5, 6, 7, 8; T, P, E, E, K in which 6 and E are highlighted. Primer 1 begins to start above the first strand from the 5 prime ends at the 5 prime ends of the strand. Primer 2 begins to start below the second strand from the 5 prime ends at the 5 prime ends of the strand. The Hb beta superscript S shows a double-stranded sequence. The sequence in the strand from 5 prime to 3 prime reads, ACT CCT GTG (T highlighted) GAG AAG. The sequence in the strand from 3 prime to 5 prime reads, TGA GGA CAC (A highlighted) CTC TTC. The above sequence reads, 4, 5, 6, 7, 8; T, P, V, E, K in which 6 and V are highlighted. Primer 1 begins to start above the first strand from the 5 prime ends at the 5 prime ends of the strand. Primer 2 begins to start below the second strand from the 5 prime ends at the 5 prime ends of the strand. Part B shows genotyping for sickle cell disease. In all three graphs, A, G, C, and T are color coded with different shades, and the respective color curve is graphed above. The colo-coded curves peak above the respective letter in the following sequence. The Hb beta superscirpt A homozygote shows ACTCCTGAGGAGAAG. The Hb beta superscirpt A or Hb beta superscript S heterozygote shows ACTCCTG (A or T) GGAGAAG. The Hb beta superscirpt S homozygote shows ACTCCTGTGGAGAAG.
Dr. Dinh Phong Son
