Clinical Chemistry
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Clinical Chemistry 45: 771-776, 1999;
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(Clinical Chemistry. 1999;45:771-776.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Optimization of Apolipoprotein(a) Genotyping with Pulsed Field Gel Electrophoresis

Pieter H. Griffioen1, Louwerens Zwang1, Ron H.N. van Schaik1, Henk Engel2, Jan Lindemans1,a and Christa M. Cobbaert3

1 University Hospital Rotterdam Dijkzigt, Department of Clinical Chemistry, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

2 St. Sophia Hospital, Department of Clinical Chemistry, P.O. Box 10400, 8000 GK Zwolle, The Netherlands.

3 Hospital De Baronie, Department of Clinical Chemistry, P.O. Box 90157, 4800 RL Breda, The Netherlands.
a Author for correspondence. Fax 31-10-4367894; e-mail lindemans{at}ckcl.azr.nl

Background: Increased lipoprotein(a) is a risk factor for atherosclerosis, and its concentration in serum is inversely correlated with the size of the apoliprotein(a) [apo(a)] component. The size of the apo(a) gene is determined mainly by the Kringle IV size polymorphism. We have optimized and characterized pulsed field gel electrophoresis (PFGE) for apo(a) genotyping.

Methods: Established PFGE protocols were adjusted. The changes included the following: (a) increased DNA yields by the use of all leukocytes for isolation from either 3 mL of fresh EDTA whole blood or 250 µL of frozen buffy coats; (b) increased efficiency of Kpn1 digestion by the inclusion of a digestion buffer wash; (c) reduction of assay time by the use of capillary blotting; (d) increased sensitivity by the use of four digoxigenin-labeled apo(a) probes; and (e) identification using a single film by the inclusion of a digoxigenin-labeled lambda marker probe in addition to apo(a) probes in the hybridization mix.

Results: In older Caucasians, 93% (buffy coats, n=468) were heterozygous for apo(a) gene size. An inverse correlation between serum lipoprotein(a) and the sum of Kringle IV alleles was found (y = -23x + 1553; r = -0.442; n = 468). Gel-to-gel variation was minimal (3%). Imprecision (SD) was one Kringle IV repeat (control sample containing eight fragments of 72–233 kb; n=34 electrophoretic runs).

Conclusions: The practicality and sensitivity of the apo(a) genotyping technique by PFGE were improved, and accuracy and reproducibility were preserved. The optimized procedure is promising for apo(a) genotyping on frozen buffy coats from large epidemiological studies.© 1999 American Association for Clinical Chemistry






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