Titre du document / Document title

Evaluation of a new pooling strategy based on leuko cyte count for rapid quantification of allele frequencies

Auteur(s) / Author(s)

ROSSMANN Heidi ⁽¹⁾ ; BÜCHLER Elena ⁽¹⁾ ; WENZEL Jürgen J. ⁽¹⁾ ; NEUKIRCH Carolin ⁽¹⁾ ; DU PREL Jean-Baptist ⁽²⁾ ; LACKNER Karl J. ⁽¹⁾ ;

Affiliation(s) du ou des auteurs / Author(s) Affiliation(s)

⁽¹⁾ Department of Clinical Chemistry and Laboratory Medicine, University of Mainz, Mainz, ALLEMAGNE
⁽²⁾ Institute of Medical Biometry, Epidemiology and Informatics (IMBEI), University of Mainz, Mainz, ALLEMAGNE

Résumé / 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. Conclusion: 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.

Revue / Journal Title

Clinical chemistry   ISSN 0009-9147   CODEN CLCHAU 

Source / Source

2007, vol. 53, n^o5, pp. 980-982 [3 page(s) (article)] (5 ref.)

Langue / Language

Anglais

Editeur / Publisher

American Association for Clinical Chemistry, Washington, DC, ETATS-UNIS  (1955) (Revue)

Mots-clés anglais / English Keywords

Molecular biology ; Clinical biology ; Biochemistry ; Gene frequency ; Quantitative analysis ; Numeration ; Evaluation ;

Mots-clés français / French Keywords

Biologie moléculaire ; Biologie clinique ; Biochimie ; Fréquence génique ; Analyse quantitative ; Numération ; Evaluation ;

Mots-clés espagnols / Spanish Keywords

Biología molecular ; Biología clínica ; Bioquímica ; Frecuencia génica ; Análisis cuantitativo ; Numeración ; Evaluación ;

Localisation / Location

INIST-CNRS, Cote INIST : 7603, 35400014939277.0250