1. A Novel 5′ (40^41insA) Mutation in a Patient with Numerous Manifestations of Cowden Disease 
Anthony M. Raizis, Martin M. Ferguson, David T. Nicholls, Derek W. Goodisson, Peter M. George 
Journal of Investigative Dermatology 
Volume 114, Issue 3, Pages (March 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Genetic analysis of a family with Cowden disease. (a) Exon 1 sequence (Genbank AF000730) analysis of the PTEN gene. Exon 1 was amplified by PCR with primers, forward 5′-TCTGCCATCTCTCTCTCCTCCT-3′ and reverse 5′-TCCGTCTACTCCCACGTTCT-3′. DNA was sequenced using the forward primer and the proband is heterozygous for an A insertion at nucleotides 40–41 (asterisk). (b) Restriction analysis of the PTEN mutation 40^41InsA. To confirm the base substitution, a restriction enzyme assay was designed using a forward mutagenic primer 5′-CAAAGAGATCGTTAGCAGCCACAA-3′. To generate a BslI restriction site ‘‘CCN7GG’’ specific for the mutation, nucleotides 33–34 ‘‘AA’’ were replaced by ‘‘CC’’ (underlined above) such that BslI cleaves the mutant (40^41InsA) allele, where the number of nucleotides between ‘‘CC’’ and ‘‘GG’’ is seven, as opposed to six in the wild-type allele. The 132 bp PCR product produced was analyzed by PAGE electrophoresis after incubation and digestion with 5 U of BslI. Genomic DNA from the mother and father was extracted from tissue obtained from paraffin blocks. BslI cleaves the mutant allele to obtain a 108 bp fragment (arrowed left). Lane 1 contains an unaffected control and other lanes are as indicated in the family tree. The proband is arrowed.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions