Figure 5.

Simultaneous detection of KIR3DL1/S1 polymorphism and copy-number using pyrosequencing. (A, top) The diploid sequence of a 17-bp fragment of exon 3 from an individual heterozygous for 3DL1*01502 and 3DS1*01301. (Gray) SNP g336a. Underneath is a pyrogram obtained from the same individual. To generate the pyrogram, nucleotides were added to a single-strand template in the sequence shown, 1–11, and correspond to peaks of the same number. The peak height is proportional to the quantity of nucleotides that were incorporated, as shown by peak 1 (the sequence is gg), which is twice the height of peak 6 (g). Each peak is a sum of the haplotypes present, so that the monomorphic positions (relative peak height = 1) are used for calibration. The combined peak height at the heterozygous position shown (peaks 10 + 11) is equal to the peak from a single monomorphic position. Shown are four different pyrograms; (top left of each diagram) the derived genotype; (brackets) the peak-height ratio compared with the single monomorphic peak. (A) At SNP g336a, there is one peak for g and one for a (peaks 10 and 11), and each peak is half the height of a single peak (peak 6). This individual has one copy of 3DL1*01502 and one copy of 3DS1*01301 (0.5g: 0.5a). (B) At SNP g336a, there is one peak for g (peak 10) that is the same height as a single monomorphic position (peak 6) and no peak on addition of nucleotide a-11. This individual is homozygous, having two copies of 3DL1*01502 (1g: 0a). (C) There are two peaks as for A, but g is twice the height of a (0.67g: 0.33a), and their sum is the same as peak 6. This individual has two copies of 3DL1*01502 and one 3DS1*01301. (D) This individual has one copy of 3DL1*01502 and two of 3DS1*01301 (0.33g: 0.67a).