QuantiLyse: reliable DNA amplification from single cells.
Amplification of DNA sequencesfrom single cells via PCR is increasingly used in basic research and clinical diagnostics but remains technically difficult. We have developed a cell lysis protocol that uses an optimized proteinase K solution, named QuantiLyse and permits reliable amplification from individual cells. This protocol was compared to other published methods by means of real-time PCR with molecular beacons. The results demonstrate that QuantiLyse treatment of single lymphocytes renders gene targets more availablefor amplification than other published proteinase K methods or lysis in water. QuantiLyse and an optimized alkaline lysis were equally effective in terms of target availability, although QuantiLyse offers greaterflexibility, as it does not require neutralization and can comprise a higher percentage of the final PCR volume. Maximum gene target availability is also obtained following QuantiLyse treatment of samples containing up to 10000 cells (the largest number tested). Thus, QuantiLyse maximizes the chances that targeted DNA sequences will be available for amplification during the first cycle of PCR, thereby reducing the variability among replicate reactions as well as the likelihood of amplification failure or allele drop-out. QuantiLyse will be useful in a range of investigations aimed at gene detection in small numbers of cells.
Pierce
Kenneth E
KE
Brandeis University, Waltham, MA 02454-9110, USA.
Rice
John E
JE
Sanchez
J Aquiles
JA
Wangh
Lawrence J
LJ
D000468:Alkalies / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology*; D006801:Humans; D006878:Hydroxides / Q000494:pharmacology; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D017680:Potassium Compounds / Q000494:pharmacology; D014867:Water
D000468:Alkalies; D003902:Detergents; D006878:Hydroxides; D017680:Potassium Compounds; D014867:Water; D019286:Endopeptidase K; D008274:Magnesium; D004229:Dithiothreitol; C029943:potassium hydroxide