Alpha thalassemia detection by gap PCR

Detection of common alpha thalassemia mutations by gap PCR.
(A) Diagram depicting the -alpha 3.7 deletion. A1, A2, and A3 show the location of the primers used in the detection of common -alpha 3.7 deletion. The deletion removes the 3' portion of the alpha 2 gene and the 5' portion of the alpha 1 gene (the area between the two diagonal lines in the top portion of the figure), resulting in a hybrid alpha2/alpha1 gene, as shown in the lower portion. This results in the amplification of a 2065 base pair fragment with primers A1 and A3 when deletion is present.
(B) Table showing the nucleotide (nt) sequence of the primers A1, A2, and A3 and their positions (location) in the alpha globin gene cluster on the reference sequence HUMHBB.
(C) Detection of the common -alpha 3.7 deletion by gap PCR. Lanes 1 and 8: DNA size markers (1 kb ladder). Lanes 2, 4, and 6 use primers (A1 and A2) for the normal alpha2 gene, while lanes 3, 5, and 7 use primers (A1 and A3) to detect the hybrid gene. Lanes 2 and 3: heterozygous -alpha 3.7 deletion; 1789 bp fragment is generated by the normal control, located on the alpha 2 gene in trans chromosome; 2065 bp fragment is generated by the deletion. Lanes 4 and 5: individual with homozygous deletion; note that there is no amplification of the 1789 bp fragment (lane 4) indicating the absence of the normal gene. Lanes 6 and 7: individual with normal alpha chains; note that there is no amplification of the 2065 bp fragment (lane 7) indicating that there is no -alpha 3.7 deletion (hence the presence of only the 1789 bp fragment in lane 6).