POLYMERASE CHAIN-REACTION AMPLIFICATION SPECIFICITY - INCIDENCE OF ALLELE DROPOUT USING DIFFERENT DNA PREPARATION METHODS FOR HETEROZYGOUS SINGLE CELLS

Purpose: The purpose was to evaluate methods of DNA preparation in a s ingle cell to determine the ability to amplify and correctly diagnose a targeted gene.Methods: One- or two-cell lymphoblasts (n = 100/group) , heterozygous for the normal and 4-base pair insertion on exon 11 of the beta-hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (L N(2)); (2) potassium hydroxide/dithiothreitol, heated to 65 degrees C, followed by acid neutralization (KOH); (3) boiling only (Bl); and (4) water only (H2O). Cells were analyzed by polymerase chain reaction us ing nested primers. Results: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozyg ous), the normal allele, or the mutant allele were as follows, respect ively: LN(2) [38], 11, 16, 11; KOH [97], 91, 5, 1; Bl [41], 17, 13, 11 ; and H2O [85], 41, 16, 28. With two cells per reaction tube the resul ts were as follows: LN(2) [85], 53, 14, 18; and KOH [97], 96, 1, 0. Co nclusions: KOH lysis was significantly greater than with all other met hods (P < 0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine t he ability to amplify both alleles as a method of quality control for single-cell analysis.