Becton Dickinson and Company Limited

Daniel L Woodard

Adriann J Howard

James A Down

Randal A Hoke

Patrick D Mize

J Michael Quante

O Elmo Millner, Deceased

Adriann H Walters

Michael C Little

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

The present invention relates to a silicon-containing material which exhibits sufficient hydrophilicity and sufficient electropositivity to bind DNA from a suspension containing DNA and permit elution of the DNA from the material. Generally, the hydrophilic and electropositive characteristics are expressed at the surface of the silicon-containing material. Preferred silicon-containing materials of the present invention include boron silicate, aluminum silicate, phosphosilicate, silica carbonyl, silica sulfonyl and silica phosphonyl. The silicon-containing materials of the present invention are particularly useful in processes for purification of DNA from other cellular components. In these processes, a suspension of cellular components is placed in contact with the silicon-containing material, the silicon-containing material is washed to remove all cellular components other than DNA which are bound to the material, and the bound DNA is eluted from the material. Several of the silicon-containing materials are capable of binding and eluting DNA using only water.

DNA purification on modified siligates

The present invention relates to modified glass fiber membranes which exhibit sufficient hydrophilicity and sufficient electropositivity to bind DNA from a suspension containing DNA and permit elution of the DNA from the membrane. Generally, the hydrophilic and electropositive characteristics are expressed at the surface of the modified glass fiber membrane. Preferred modified glass fiber membranes of the present invention include glass fiber membranes that have been modified by treatment with trifluoroacetic acid (TFA), BCl.sub.3, SiCl.sub.4, NaOH, F.sup.-, AlCl.sub.3 alone or in combination, with or without water. The modified glass fiber membranes of the present invention are particularly useful in processes for purification of DNA from other cellular components. In these processes, a suspension of cellular components is placed in contact with the modified glass fiber membrane, the modified glass fiber membrane is washed to remove all cellular components other than DNA which are bound to the membrane, and the bound DNA is eluted from the membrane.

Modified glass fiber membranes useful for DNA purification by solid

The present invention relates to fluorinated surfaces which exhibit sufficient hydrophilicity and sufficient electropositivity to bind DNA from a suspension containing DNA and permit elution of the DNA from the surface. Generally, the hydrophilic and electropositive characteristics are expressed at the fluorinated surface. Preferred fluorinated surfaces of the present invention include fluorinated Al(OH).sub.3, fluorinated SiO.sub.2 and fluorinated Celite. The fluorinated surfaces of the present invention are particularly useful in processes for purification of DNA from other cellular components. In these processes, a suspension of cellular components is placed in contact with the fluorinated surface, the fluorinated surface is washed to remove all cellular components other than DNA which are bound to the surface, and the bound DNA is eluted from the surface. Lower concentrations of chaotrope in the binding buffer are needed to bind DNA to the fluorinated surfaces.

DNA purification by solid phase extraction using fluorinated celite

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

DNA purification by solid phase extraction using a hydroxide-washed

DNA purification by solid phase extraction using glass fiber membrane

Glass fiber membranes modified by treatment with SiCl.sub.4, AlCl.sub.3

DNA purification by solid phase extraction using trifluorometric acid

Chromogenic and fluorogenic substrates for .beta.-lactamase, methods for synthesis thereof and methods for detecting .beta.-lactamase in a sample are provided. The substrates are substantially colorless or substantially nonfluorescent .beta.-lactam compounds which include an electronegative leaving group. The leaving group comprises a carbamate, carbonate, thiocarbamate or thiocarbonate linkage and a fluorescent moiety or a moiety capable of producing a visually detectable colored product. Upon cleavage of the lactam ring by .beta.-lactamase, the leaving group is liberated and fluorescence or a colored product is produced.

Fluorogenic and chromogenic .beta.-lactamase substrates

Silicon tetrahydrazide compounds produced by reacting SiCl.sub.4 with hydrazine to completion. The compounds are useful for binding DNA, as they do not require the use of chaotrope or alcohol solutions for binding. Binding and elution of DNA can be accomplished in low salt buffers or water. Kits containing the compounds for purification of DNA are also disclosed.

Silicon tetrahydrazide for purification of DNA

Boron silicates, phosphosilicates and aluminum silicates useful as binding surfaces for DNA purification. These compounds allow DNA to be bound and eluted under native conditions (i.e., in the absence of chaotropes or alcohols) using only water, low salt buffers or physiological buffers. Methods for preparation of the compounds and methods for purifying DNA using the compounds are also provided.

Boron silicates, aluminum silicates, and phosphosilicates for

A process for purifying DNA comprising binding the DNA to a silicate compound in the presence of water, low salt buffer, or physiological buffer and eluting in the bound DNA by heating in a low salt buffer, a physiological buffer, or water. The silicate compound is prepared by reacting SiCl.sub.4 with a molar percentage of BCl.sub.3 of about 33.5%, 55.6%, or 83.3%, with a molar percentage of PCl.sub.3 of about 50.0%-83.3%, or with a molar percentage of AlCl.sub.3 of about 9.1%; cooling the reaction mixture; adding water until the evolution of gas is complete; and recovering the compound.

Purification of DNA on aluminum silicates, and phosphosilicates

A process for preparing borosilicate, aluminosilicate, or phosphosilicate comprising preparing a mixture of SiCl.sub.4 with a specific molar ratio of BCl.sub.3, AlCl.sub.3, or PCl.sub.3 ; cooling said mixture to zero degrees; and adding water to the reaction mixture until the evolution of gas is complete. These silicates are useful for purifying DNA wherein the DNA is bound in water or low salt buffers (i.e., in the absence of a chaotropic agent or alcohol).

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Raleigh, NC, United States of America

Daniel L Woodard