title: Xeroderma pigmentosum variant: complementary molecular approaches to detect a 13 base pair deletion in the DNA polymerase eta gene.
abstract: Deficiencies of DNA polymerase eta-an enzyme mediating replication past UV-induced DNA damage-predispose individuals to xeroderma pigmentosum variant (XPV) and result in a high incidence of skin cancers. We designed, developed and assessed several complementary molecular approaches to detect a genetically inherited deletion within DNA polymerase eta. RNA was reverse transcribed from XPV fibroblasts and from normal human cells, and standard polymerase chain reaction (PCR) was conducted on the cDNA targeting a region with a 13 base pair deletion within the polymerase eta gene. PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis and cycle DNA sequencing. The deletion was found to eliminate a BsrGI restriction site and affected the number of resultant fragments visualized after gel electrophoresis. Cycle sequencing of polymerase eta-specific amplicons from XPV and normal cells provided a second approach for detecting the mutation. Additionally, the use of a fluorescent nucleic acid dye-EvaGreen-in real-time PCR and melt curve analysis distinguished normal and XPV patient-derived amplicons as well as heteroduplexes that represent heterozygotic carriers without the need for high resolution melt analysis-compatible software. Our approaches are easily adaptable by diagnostic laboratories that screen for or verify genetically inherited disorders and identify carriers of a defective gene.
authors.affiliation: Department of Medical Laboratory and Radiation Sciences, College of Health Sciences, Old Dominion University, Norfolk, VA 23529, USA. phentosh@odu.edu
authors.forename: Patricia
authors.initials: P
authors.lastname: Hentosh
authors.forename: Tirania
authors.initials: T
authors.lastname: Benjamin
authors.forename: Lavinia
authors.initials: L
authors.lastname: Hall
authors.forename: Shannon
authors.initials: S
authors.lastname: Leap
authors.forename: Jessica
authors.initials: J
authors.lastname: Loescher
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Poyner
authors.forename: Tabetha
authors.initials: T
authors.lastname: Sundin
authors.forename: Mary
authors.initials: M
authors.lastname: Whittle
authors.forename: Sandra
authors.initials: S
authors.lastname: Wilkinson
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Peffley
mesh_terms: D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation / Q000235:genetics; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D011467:Prostate / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048348:Reverse Transcription / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics*; D014983:Xeroderma Pigmentosum / Q000201:enzymology* / Q000235:genetics*; D000099270:Y-Family DNA Polymerases
chemical_list: D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases