Becton Dickinson and Company Limited

Melinda S Fraiser

George T Walker

James A Down

Michael C Little

G Terrance Walker

Catherine A Spargo

Mark Van Cleve

James L Schram

David J Wright

David James Wright

A process for lysing Mycobacteria to liberate nucleic acids such as DNA and RNA comprises heating the Mycobacteria in an aqueous solution to lyse the Mycobacteria and release the nucleic acids, with the aqueous solution containing a chelating agent such as EDTA or EGTA in an amount effective to inhibit degradation of the released nucleic acids. Examples of Mycobacteria which can be lysed include Mycobacterium fortuitum and Mycobacterium tuberculosis. After lysis, the nucleic acid is preferably then amplified and detected. Kits useful for carrying out the present invention are also disclosed.

Process for lysing mycobacteria

Strand Displacement Amplification methods (thermophilic SDA) which can be performed over a broad temperature range (37.degree. C. to 70.degree. C.). The preferred temperature range for thermophilic SDA is 50.degree. C. to 70.degree. C. It has been found that certain thermophilic restriction endonucleases are capable of nicking the hemimodified restriction endonuclease recognition/cleavage site as required by SDA and dissociating from the site. It has further been found that certain thermophilic polymerases are capable of extending from the nick while displacing the downstream strand. Thermophilic SDA, because of reaction temperatures higher than previously possible with conventional SDA enzyme systems, has improved specificity and efficiency, reduced nonspecific background amplification, and potentially improved yields of amplification products. In addition, the need to add the enzymes in a separate step after the initial heat denaturation of double stranded targets is eliminated when enzymes capable of tolerating the denaturation temperature are used.

Strand displacement amplification using thermophilic enzymes

.alpha.-Boronated deoxynucleoside triphosphates, when incorporated into a double-stranded restriction endonuclease recognition/cleavage site for a restriction endonuclease, induce nicking by the restriction endonuclease. .alpha.-Boronated deoxynucleoside triphosphates (dNTP.alpha.BH.sub.3) are therefore useful as nucleotide analogs in SDA to produce the nickable hemimodified restriction endonuclease recognition/cleavage site required to sustain the amplification reaction.

Nicking of DNA using boronated nucleotides

Methods for inactivating contaminating amplicons in isothermal nucleic acid amplification reactions such as SDA. Uracil is incorporated into the amplicons produced by amplification in the place of thymine (T) using novel SDA reaction conditions. If these amplicons contaminate a subsequent amplification reaction, they may be inactivated as templates (i.e., rendered unamplifiable) by treatment with UDG. As isothermal amplification does not involve elevated temperatures, the UDG may be inactivated during the subsequent amplification of specific target sequences by inclusion of the UDG inhibitor protein Ugi. Incorporation of dU has unexpectedly been found to enhance the amplification power of SDA as compared to conventional SDA reactions. The methods may also be used to detect UDG activity in reagents or samples.

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Durham, NC, United States of America

Melinda S Fraiser