Adapting in situ polymerase chain reaction for genotyping of cells in suspension.
An approach is described for in situ polymerase chain reaction (ISPCR) based on cycling primed in situ synthesis (PRINS) conditions defined for alpha-satellite DNA. Using blood cell preparations subjected to limited fixation with paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of paired nuclear spots corresponding to specific centromeres. Using longer cycling regimens, primers for single copy genes also generated paired nuclear spots in a primer-pair--specific manner. In this context, the amplification refractory mutation system (ARMS) was evaluated for in situ applications. In ARMS, allele-specific primers are used in such a manner that PCR proceeds only when an exact 3' match between annealed primer and template is recognized by DNA polymerase. Using normal and mutant primers for the delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extraction and reamplification with ARMS primers, it was shown that amplicon accumulates in cells in the expected primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that nonspecific signal is due to primer dimer formation, especially in the absence of true template DNA. The impact of primer dimer formation in generating a false-positive signal is discussed. The method described here enables a cell population to be analyzed for a given point mutation.
Brownie
J
J
Zeneca Diagnostics, Northwich, Cheshire, England.
Paskins
L
L
Little
S
S
Schwarz
M J
MJ
Bull
J H
JH
D002469:Cell Separation; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000737:chemistry; D019254:Genes, bcl-2 / Q000235:genetics*; D005838:Genotype; D006801:Humans; D017403:In Situ Hybridization; D007962:Leukocytes / Q000737:chemistry*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation