Non-isotopic silver-stained SSCP is more sensitive than automated direct sequencing for the detection of PTEN mutations in a mixture of DNA extracted from normal and tumor cells.
The sensitivity of non-isotopic PCR-SSCP was compared to direct sequencing of PTEN exons. DNA from leukocytes derived from healthy donors, and from the glioblastoma cell line LN319 was extracted and mixed in different proportions from 0 to 100%. The LN319 cell line contains a point mutation at codon 15 exon 1 of the PTEN gene. The PCR-SSCP experiments demonstrated mutations in samples containing as little as 10% tumor DNA. In contrast, direct DNA sequencing experiments were less sensitive, requiring 30-70% of tumor DNA in the sample, depending on the DNA strand sequenced. In conclusion, PCR-SSCP, in our hands, is more sensitive than automated sequencing for detecting PTEN point mutations. We recommend to always sequence both strands, and take into account that samples containing less than 30% tumor cells should not only be sequenced, but also studied by PCR-SSCP in order to discriminate false negative results.
Fan
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X
Department of Genetics, University of Navarre, E-31008 Pamplona, Spain.
Furnari
F B
FB
Cavenee
W K
WK
Castresana
J S
JS
D001932:Brain Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D009154:Mutation; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D016622:Silver Staining / Q000379:methods; D013030:Spain; D025521:Tumor Suppressor Proteins
D004273:DNA, Neoplasm; D025521:Tumor Suppressor Proteins; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human