Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.
Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.
Liu
W
W
Department of Laboratory Medicine and Pathology, Division of Experimental Pathology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Smith
D I
DI
Rechtzigel
K J
KJ
Thibodeau
S N
SN
James
C D
CD
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D029721:Drosophila Proteins; D005069:Evaluation Studies as Topic; D005091:Exons; D018095:Germ-Line Mutation; D005910:Glioma / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D025521:Tumor Suppressor Proteins
C505032:CFTR protein, human; D004273:DNA, Neoplasm; D029721:Drosophila Proteins; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; D010744:Phosphoric Monoester Hydrolases; D017027:Protein Tyrosine Phosphatases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human