Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.
Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.
Maurisse
Rosalie
R
Research Institute, California Pacific Medical Center, San Francisco, CA 94107, USA.
Fichou
Yann
Y
De Semir
David
D
Cheung
Judy
J
Ferec
Claude
C
Gruenert
Dieter C
DC
D001483:Base Sequence; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D005785:Gene Conversion; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012313:RNA; D004247:DNA