US 7,547,510 B2Granted

Thermostable proteinases from thermophilic bacteria

AssigneeZyGEM Corporation Limited

InventorsRoy McIver Daniel, David James Saul

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Abstract

The present invention relates to thermostable proteinases from thermophilic Bacillus species and their uses in the preparation of nucleic acid samples. The enzymes of the invention are stable and active at 65-80° C., but are readily autolysed or denatured above 90° C.

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Claims7

Dependent claims

A method for amplifying deoxyribonucleic acid (DNA) for testing, comprising:

i) adding Bacillus EA1 thermophilic proteinase to a sample comprising deoxyribonucleic nucleic acid (DNA) for testing, thereby forming a system, wherein the thermophilic proteinase digests protein in the sample and is capable of auto-catalysis at or above 90° C.;
ii) closing the system;
iii) incubating said sample in said system for a period at 65-80° C. as required to effect one or more of:
lysis of one or more cells, digestion of one or more proteins, or digestion of one or more cell-wall enzymes, via activity of said thermophilic proteinase;
iv) incubating the sample in said system at or above 90° C. to effect auto-catalysis of the thermophilic proteinase;
v) opening the system;
vi) providing polymerase chain reaction (PCR) reagents to the sample;
and
vii) amplifying the DNA for testing by PCR,
wherein the protein and thermophilic proteinase are denatured in the system prior to opening without the addition of further denaturing agents.

7 more elements

Dependencies are inferred from the claim text.

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Abstract

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Claims7

Dependent claims

A method for amplifying deoxyribonucleic acid (DNA) for testing, comprising:

i) adding Bacillus EA1 thermophilic proteinase to a sample comprising deoxyribonucleic nucleic acid (DNA) for testing, thereby forming a system, wherein the thermophilic proteinase digests protein in the sample and is capable of auto-catalysis at or above 90° C.;
ii) closing the system;
iii) incubating said sample in said system for a period at 65-80° C. as required to effect one or more of:
lysis of one or more cells, digestion of one or more proteins, or digestion of one or more cell-wall enzymes, via activity of said thermophilic proteinase;
iv) incubating the sample in said system at or above 90° C. to effect auto-catalysis of the thermophilic proteinase;
v) opening the system;
vi) providing polymerase chain reaction (PCR) reagents to the sample;
and
vii) amplifying the DNA for testing by PCR,
wherein the protein and thermophilic proteinase are denatured in the system prior to opening without the addition of further denaturing agents.

7 more elements

Dependencies are inferred from the claim text.

Description3,990 words

Summary3,110 words

#This invention relates to improved procedures for molecular biology techniques. In particular, it is envisaged the procedure will facilitate improved molecular biology diagnostic techniques by providing means of removing extraneous contamination, as well as simplifying the overall procedures.

#Preferably the contamination that is removed as a consequence of the present invention includes proteins and nucleases.

#Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology. It is a rapid, inexpensive and simple means of producing relatively large numbers of copies of DNA molecules (via enzymatic amplification of a specific nucleic acid sequence of interest) from minute quantities of source material, even when the source nucleic acid is of relatively poor quality.

Brief description of the drawings2 figures

#Further aspects of the present invention will become apparent from the following description, given by way of example only and with reference to the accompanying drawings in which:

#FIG. 1 is a comparison of the steps involved in DNA extraction using a standard protocol and the new protocol using proteinases in accordance with one preferred embodiment of the present invention; and

#FIG. 2 is a diagrammatic representation of the activities occurring at different temperatures using the new DNA extraction protocol in accordance with one preferred embodiment of the present invention.

Detailed description790 words

#With reference to the figures by way of example only, there is provided a method for the preparation of DNA samples for polymerase chain reaction which removes contaminating proteins and inhibitors from samples, and provides an alternative to existing procedures.

#FIG. 1 illustrates the difference in the number of steps, time taken and likely introduction of contaminants between standard protocols and the improved protocol of the present invention.

#The method of the present invention also preferably includes the addition of at least one mesophilic enzyme and at least one non-specific thermophilic enzyme to a sample containing DNA for testing and amplification via PCR.

Abstract

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Claims7

Abstract

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Claims7

Description3,990 words

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Classification3 codes

Life of this patent

Description3,990 words