title: Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats.
abstract: Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30,000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.
authors.affiliation: Department of Immunology, Erasmus MC, Rotterdam, The Netherlands. m.vanderburg@erasmusmc.nl
authors.forename: M
authors.initials: M
authors.lastname: van der Burg
authors.forename: H
authors.initials: H
authors.lastname: Kreyenberg
authors.forename: A
authors.initials: A
authors.lastname: Willasch
authors.forename: B H
authors.initials: BH
authors.lastname: Barendregt
authors.forename: S
authors.initials: S
authors.lastname: Preuner
authors.forename: F
authors.initials: F
authors.lastname: Watzinger
authors.forename: T
authors.initials: T
authors.lastname: Lion
authors.forename: E
authors.initials: E
authors.lastname: Roosnek
authors.forename: J
authors.initials: J
authors.lastname: Harvey
authors.forename: M
authors.initials: M
authors.lastname: Alcoceba
authors.forename: M G
authors.initials: MG
authors.lastname: Díaz
authors.forename: P
authors.initials: P
authors.lastname: Bader
authors.forename: J J M
authors.initials: JJ
authors.lastname: van Dongen
mesh_terms: D046528:Chimerism; D004247:DNA / Q000235:genetics*; D006580:Genetic Carrier Screening; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D012709:Serum Albumin / Q000235:genetics; D000075462:Serum Albumin, Human; D033581:Stem Cell Transplantation / Q000592:standards*; D020080:Tandem Repeat Sequences / Q000235:genetics*; D014019:Tissue Donors
chemical_list: C422455:ALB protein, human; D005819:Genetic Markers; D012709:Serum Albumin; D004247:DNA; D000075462:Serum Albumin, Human