title: Evaluation of a new pooling strategy based on leukocyte count for rapid quantification of allele frequencies.
abstract: BACKGROUND
  Allele frequencies of single-nucleotide polymorphisms (SNPs) can be quantified from DNA pools. The conventional preparation of DNA pools requires DNA isolation and quantification for each blood sample. We hypothesized that pooling of whole blood samples according to their leukocyte count, which determines DNA content, would be as reliable as the conventional pooling method but much less tedious to perform.
  
  
  METHODS
  We collected 100 whole blood samples and measured the leukocyte count. Samples were frozen until further use. After thawing, pools were generated by combining aliquots containing an equal number of leukocytes. In parallel, DNA was extracted from another aliquot, DNA concentration was measured, and DNA concentration-based pools were assembled. All original samples were genotyped directly using 4 different SNP assays to obtain the exact allele frequencies in the pool. In addition, samples of known genotypes were mixed according to the DNA concentration or the leukocyte count to generate artificial samples of known allele frequencies. We analyzed pools and mixes in triplicate by pyrosequencing and calculated allelic frequencies.
  
  
  RESULTS
  Leukocyte and DNA pooling provided equally accurate and precise SNP frequencies comparable to published data.
  
  
  CONCLUSIONS
  DNA and leukocyte pooling are both suitable strategies to determine allele frequencies in frozen samples. The leukocyte pooling approach is much less tedious, quicker, and less expensive. It should be always considered if leukocyte counts are available.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, University of Mainz, Mainz, Germany. rossmann@zentrallabor.klinik.uni-mainz.de
authors.forename: Heidi
authors.initials: H
authors.lastname: Rossmann
authors.forename: Elena
authors.initials: E
authors.lastname: Büchler
authors.forename: Jürgen J
authors.initials: JJ
authors.lastname: Wenzel
authors.forename: Carolin
authors.initials: C
authors.lastname: Neukirch
authors.forename: Jean-Baptist
authors.initials: JB
authors.lastname: du Prel
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Lackner
mesh_terms: D001322:Autoanalysis; D004247:DNA / Q000097:blood / Q000235:genetics*; D005787:Gene Frequency; D006801:Humans; D007958:Leukocyte Count; D007962:Leukocytes / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide
chemical_list: D004247:DNA