Genotyping: a new application for the spent dialysate in peritoneal dialysis.
BACKGROUND
The dialysate of patients on peritoneal dialysis (PD) is used to determine the concentration of growth factors and cytokines, and as a source of resident peritoneal cells for subsequent culture experiments. We hypothesized that the cells contained in spent dialysate samples obtained at the time of the peritoneal equilibration test (PET) and subsequently stored may represent a source of DNA from a given PD patient.


METHODS
We characterized a protocol of DNA extraction from dialysates obtained in PD patients after a long dwell during the initial PET and kept frozen up to several years. After amplification of the source DNA by strand displacement using the bacteriophage phi 29 DNA polymerase, we performed polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for the Glu298Asp polymorphism of ENOS to demonstrate the suitability of the extracted DNA for genotyping.


RESULTS
A significant amount of DNA (mean yield 12 microg/ml dialysate) was extracted from frozen dialysate samples. The extraction yield was not influenced by the duration of storage at -20 degrees C. Following amplification, the DNA extracted from the dialysate was used successfully for genotyping the Glu298Asp polymorphism of ENOS, as demonstrated by parallel analyses using DNA extracted from the peripheral blood and sequencing.


CONCLUSIONS
These results demonstrate that the dialysis effluent obtained at the time of the initial PET and stored at -20 degrees C is a reliable source of DNA that can be used subsequently for PCR amplification, RFLP analysis and sequencing.
Division of Nephrology, Université Catholique de Louvain Medical School, Brussels, Belgium.
Gaëlle
G
Gillerot
Huguette
H
Debaix
Olivier
O
Devuyst
D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D015314:Dialysis Solutions / Q000032:analysis*; D005838:Genotype; D006801:Humans; D010529:Peritoneal Cavity; D010530:Peritoneal Dialysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D017931:DNA Primers; D015314:Dialysis Solutions; D004247:DNA