Interference of heparin with the polymerase chain reaction.

Author(s): Beutler E, Gelbart T, Kuhl W

Publication: Biotechniques, 1990, Vol. 9, Page 166

PubMed ID: 2400599 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of type of anticoagulant and refrigerated storage on the amplificability of DNA extracted from whole blood. Methods to purify DNA from residual heparin were also investigated. Extraction was completed using the Applied Biosystems 340A nucleic acid extractor for unfractionated blood.

   Summary of Findings:

   Without additional measures taken to remove residual heparin, a fragment of the glucocerebrosidase gene could not be amplified from DNA extracted from either fresh or refrigerated blood specimens collected into tubes containing sodium heparin. The glucocerebrosidase gene fragment was amplified from fresh or refrigerated specimens collected into tubes containing EDTA. Separation of the leukocytes by centrifugation or sedimentation and washing the white blood cells in saline prior to DNA extraction, washing whole blood cells prior to DNA extraction, or treating the DNA with heparinase II enabled amplification of the glucocerebrosidase gene fragment from heparinized specimens. The authors state that boiling DNA along with filtration on Sephadex G-75, altering the pH followed by gel filtration, repeated ethanol precipitation, or titration with protamine sulfate did not allow amplification of the glucocerebrosidase gene fragment from heparinated specimens.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	EDTA Sodium heparin
   Storage	Storage duration	0 d 10 d
   Analyte Extraction and Purification	Analyte purification	Heparinase II treatment No heparinase II treatment Boiling DNA No boiling of DNA Acidification of DNA Alkalinization of DNA Repeated ethanol precipitation Titration with protamine sulfate
   Biospecimen Aliquots and Components	Blood and blood products	Leukocyte Whole blood
   Analyte Extraction and Purification	Filtration of purified DNA	No Yes Gel Sephadex
   Biospecimen Aliquots and Components	Blood processing method	Washed whole blood cells Washed white blood cells Unwashed blood
   PCR Specific	Targeted nucleic acid	Glucocerebrosidase

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