Becton Dickinson and Company Limited

Other

Adriann H Walters

James A Down

Daniel L Woodard

Michael C Little

William E Keating

Deborah R Howard

Margaret S Dey

Daretta A Bruchey

The present invention relates to a sample processing method for rendering cellular components such as nucleic acids in a sample accessible. The method involves subjecting a sample of disrupted cells to sufficient agitation in the presence of particles to separate nucleic acids from other cellular components. A heat lysis process without other lysogenic agents or conditions is the preferred method of disrupting cells for the sample. Once accessed, the nucleic acids may be used in various molecular biology procedures.

Method to access nucleic acids from cells

Compositions and processes for isolation or purification of DNA are provided. The compositions are hydroxylated silica polymers produced by reacting silicon dioxide with an alkaline solution, followed by acidification. The hydroxylated silicas produced by this process can be used to bind DNA in aqueous solutions, without the need for binding reagents such as alcohols or chaotropes. The bound DNA may then be separated from the solution and eluted into water or a buffer by heating.

Purification of DNA on Hydroxylated Silicas

It has been found that certain glycoproteins, particularly mucins, are inhibitors of nucleic acid amplification reactions and that inhibition of the amplification reaction is associated with partial degradation of the carbohydrate chain. Partial degradation of the carbohydrate of a non-inhibitory glycoprotein renders it inhibitory, and partial degradation of the carbohydrate of a slightly inhibitory glycoprotein makes it more inhibitory. Sample processing prior to amplification may contribute to partial degradation of the carbohydrate chains of the glycoproteins which are present and increase their inhhibitory effect. In contrast, complete removal of the carbohydrate significantly reduces or completely eliminates the inhibitory effect. Methods for reducing or eliminating glycoprotein-associated inhibition of nucleic acid amplification reactions are also disclosed.

Methods for reducing inhibition of nucleic acid amplification reactions

Methods for processing samples which may contain mycobacteria which are compatible with both conventional culturing techniques and nucleic acid analysis. It has been found that the harsh chemical treatment previously thought necessary to obtain efficient lysis can be eliminated without loss of lysis efficiency, thereby eliminating reagents which may inhibit subsequent nucleic acid-based reactions. Heat alone is sufficient to lyse mycobacteria, even in buffers which do not contain detergents, chelators, enzymes, etc. Residual reagents introduced by conventional sample processing methods for culture can be successfully removed from the processed pellet without significant loss of microorganisms by at least two washings with saline, water, or buffers compatible with nucleic acid analysis and appropriate centrifugation.

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Baltimore, MD, United States of America

Adriann H Walters