Polymerase chain reaction amplification specificity: incidence of allele dropout using different DNA preparation methods for heterozygous single cells.
OBJECTIVE
The purpose was to evaluate methods of DNA preparation in a single 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 (LN2); (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 using nested primers.


RESULTS
The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN2 [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 results were as follows: LN2 [85], 53, 14, 18; and KOH [97], 96, 1, 0.


CONCLUSIONS
KOH lysis was significantly greater than with all other methods (P < 0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine the ability to amplify both alleles as a method of quality control for single-cell analysis.
Gitlin
S A
SA
Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA. sueg@jones1.evms.edu
Lanzendorf
S E
SE
Gibbons
W E
WE
D000483:Alleles; D016477:Artifacts; D002458:Cell Fractionation / Q000379:methods*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004252:DNA Mutational Analysis / Q000379:methods*; D004229:Dithiothreitol / Q000494:pharmacology; D005091:Exons / Q000235:genetics; D005240:Feasibility Studies; D005615:Freezing; D006579:Heterozygote; D006358:Hot Temperature; D006801:Humans; D006878:Hydroxides / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics*; D008214:Lymphocytes / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D008832:Microchemistry; D009584:Nitrogen; D016133:Polymerase Chain Reaction / Q000379:methods*; D017680:Potassium Compounds / Q000494:pharmacology; D011237:Predictive Value of Tests; D011296:Prenatal Diagnosis / Q000379:methods*; D012680:Sensitivity and Specificity; D013661:Tay-Sachs Disease / Q000175:diagnosis* / Q000201:enzymology / Q000235:genetics / Q000517:prevention & control; D014867:Water / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
D006878:Hydroxides; D007527:Isoenzymes; D017680:Potassium Compounds; D014867:Water; D004247:DNA; D001619:beta-N-Acetylhexosaminidases; D009584:Nitrogen; D004229:Dithiothreitol; C029943:potassium hydroxide