title: Allelic loss analysis by denaturing high-performance liquid chromatography and electrospray ionization mass spectrometry.
abstract: Analysis of allelic imbalance is of great importance for understanding tumorigenesis and the clinical management of malignant disease. Fluorescent-based capillary electrophoresis (CE) of highly polymorphic short tandem repeats (STRs) has become the main method used to detect the loss/gain of alleles. However, there is continued interest in the development of techniques that require no fluorescence and allow the rapid analysis of individual samples. One promising alternative is ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), which is widely available because of its use in denaturing HPLC. Its applicability in combination with ultraviolet (UV) absorbance detection to the efficient separation of di- and tetranucleotide repeats on the short arm of chromosome 11 was tested using 25 matched pairs of normal and ovarian cancer tissues. Loss of heterozygosity (LOH) could be readily identified for all 13 loci tested, based on changes in the ratios between either the alleles or homo- and heteroduplex signals. However, discrimination between noninformative homo- or hemizygous and heterozygous samples was difficult or impossible when HPLC failed to resolve the alleles. Hyphenation of HPLC with electrospray ionization (ESI) quadrupole ion trap (IT) mass spectrometry (MS) not only allowed the identification of coeluting alleles, but also the reliable detection of a 40% reduction of one allele. The size range of DNA fragments amenable to mass spectrometric analysis was effectively tripled to >300 bp by the use of a linear IT and a Taq DNA polymerase cocktail lacking detergents that otherwise adversely affect ESI.
authors.affiliation: Department of Obstetrics and Gynaecology, Technical University, Munich, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Gross
authors.forename: Georg
authors.initials: G
authors.lastname: Hölzl
authors.forename: Norbert
authors.initials: N
authors.lastname: Arnold
authors.forename: Evelyn
authors.initials: E
authors.lastname: Hauenstein
authors.forename: Anja
authors.initials: A
authors.lastname: Jacobsen
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schulze
authors.forename: Juliane
authors.initials: J
authors.lastname: Ramser
authors.forename: Alfons
authors.initials: A
authors.lastname: Meindl
authors.forename: Marion
authors.initials: M
authors.lastname: Kiechle
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Oefner
mesh_terms: D002277:Carcinoma / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats; D010051:Ovarian Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019914:Taq Polymerase / Q000032:analysis
chemical_list: D019914:Taq Polymerase