WO2002000930A2 - Method for dna extraction - Google Patents
Method for dna extraction Download PDFInfo
- Publication number
- WO2002000930A2 WO2002000930A2 PCT/GB2001/002909 GB0102909W WO0200930A2 WO 2002000930 A2 WO2002000930 A2 WO 2002000930A2 GB 0102909 W GB0102909 W GB 0102909W WO 0200930 A2 WO0200930 A2 WO 0200930A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sample
- dna
- retaining support
- screening
- extraction
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- the present invention provides an improved method of DNA extraction from a dried faecal sample. More specifically, the invention provides a method for extracting DNA from dried faecal smears such that the DNA can be accurately screened for mutations, epigenetic alterations, and other markers specific for colorectal cancer and further for a wide range of other diseases and conditions including veterinary applications.
- a dried faecal smear is rehydrated and a chemical reaction with hydrogen peroxide allows the detection of small quantities of blood invisible to the naked eye.
- the drawback of detection of occult blood on a dried faecal smear is that it is not specific for gastrointestinal cancer (especially colorectal cancer) and its sensitivity is poor. Thus many false positives are possible (with the associated drawbacks of patient anxiety, and consequent exposure to investigation such as colonoscopy, which carries significant mortality) , as well as false negatives, where patients with carcinoma are not correctly diagnosed and not subject to further investigation or treatment.
- the present invention provides a method of extracting DNA from faeces comprising; smearing a small amount of the faeces onto a sample retaining support, allowing the smeared sample to dry on the retaining support, storing the sample on the sample retaining support, and extracting DNA from the sample using a standard DNA extraction procedure.
- the sample retaining support is cardboard or paper.
- sample retaining support is guaiac-impregnated card.
- sample retaining support is nylon
- the present invention further provides a method of extracting DNA from dried faeces such that PCR can be used to determine the presence of genetic mutations / markers associated with specific diseases or conditions, comprising;
- a further aspect of the present invention provides a method of extracting from a faecal sample obtained from an animal, for analysis of the presence of genetic markers specific for disease, comprising the steps of, smearing a small amount of faecal sample onto a sample retaining support, drying the sample, extracting DNA from the sample by means of a standard DNA extraction procedure and analysing the DNA by means of PCR for the presence of genetic markers indicative of disease.
- Extraction of DNA from the dried faecal sample may be by any suitable technique present in the art, wherein the DNA extraction process results in there being no interference from dietary components present in smear and there being no interference from bacterial DNA present in the sample.
- DNA extraction is based on the guanidinium extraction method published by Karlinsey J. et al (Simultaneous purification of DNA and RNA from small numbers of eukayotic cells) Anal Biochem. 1989. Aug 1. 180(2) p 303-306.
- the DNA can be extracted by commercially available kits, such as the Quiagen DNA extraction kit. Examination of the extracted DNA may be by screening for any suitable tumour or cell proliferation DNA marker .
- any suitable mutation or marker specific for colorectal cancer or cell dysplasia may be chosen.
- Preferably screening of the extracted DNA for suitable mutations or markers is by PCR, wherein primers specific for the amplification of the area of DNA where the mutation may occur is effected.
- the present invention shows that if a faecal sample is smeared onto a suitable matrix such as card, paper or nylon the sample can be stored on this card for an indefinite period of time and still permit the extraction of DNA from the sample, which can be used in analysis.
- a suitable matrix such as card, paper or nylon
- the method of the present invention allows extraction of DNA of a quality comparable to other more laborious, u'npleasant and expensive methods and thus is particularly advantageous due to its economy of use, convenience and further due to the stability of the sample when smeared on the card in that it exhibits long term stability even at room temperature i.e. no refrigeration or specific sample storage conditions are required.
- sample collection is a much simplified and pleasant procedure for the patient, especially to those who may be considered to be in a high risk cancer group and thus require frequent screening.
- the first step of the analytical procedure is the extraction of the DNA from the sample using the guanidinium based technique; the area of card smeared with the faecal sample is shredded and then rehydrated in guanidinium isothocyanate buffer. After incubation for rehydration, the sample is vortexed to disrupt the card. Further to spinning out the pellet debris, the supernatant is extracted several times with glass milk solution to bind the DNA. DNA is released into solution from the silica matrix by treatment with a low-salt buffer. The DNA sample is suitable for analysis by PCR.
- An alternative method of extracting DNA from the dried faecal sample would comprise the steps of shredding the area of card smeared with the faecal sample and rehydrating this in guanidinium isothocyanate buffer, as in the first method.
- the liquid generated is then treated with proteinase K.
- This mixture is then extracted twice with phenol/chloroform, and finally once with chloroform.
- the resulting supernatant is then treated with ice cold absolute alcohol to precipitate the DNA, which can be amplified by PCR for analysis.
- DNA can be extracted using commercially available kits such as the Qiagen DNA extraction kit .
- the DNA can subsequently be analysed by PCR.
- PCR Analysis by PCR would involve primers which can be used to amplify and thus show the presence of DNA normal to human or alternatively DNA analogous to tumours.
- the present technique therefore allows for the sensitive and accurate extraction and analysis of DNA from faecal samples, wherein the sample can be simply and hygienically transported by the postal system to a laboratory and has the following advantages over tests previously known in the art:
- tumour or cell proliferation DNA markers may be studied.
- markers specific for colorectal cancer cell dysplasia or neoplasia may be chosen.
- the present invention does not suffer from interference from dietary components and further specificity is greatly improved.
- DNA specific for the presence of neoplasia / cancer can be identified from patient samples, (iii)DNA markers or mutations detected in the card should correlate with patient tumour or polyp samples,
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- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
There is provided a method of extracting both human specific and tumour specific DNA from dried faecal smears. This method allows the screening of the extracted DNA for genetic mutations or markers associated with specific diseases. In particular this method m be used to test for gastrointestinal cancer and more particularly colorectal cancer. In addition to allowing the sensitive and accurate analysis of samples this method enables simple and hygienic transport of samples by postal system.
Description
nDNA Extraction"
The present invention provides an improved method of DNA extraction from a dried faecal sample. More specifically, the invention provides a method for extracting DNA from dried faecal smears such that the DNA can be accurately screened for mutations, epigenetic alterations, and other markers specific for colorectal cancer and further for a wide range of other diseases and conditions including veterinary applications.
Current technologies used for the screening of colorectal cancer include occult blood screening, colonoscopy and examination of DNA in fresh stool .
In occult blood screening, a dried faecal smear is rehydrated and a chemical reaction with hydrogen peroxide allows the detection of small quantities of blood invisible to the naked eye. The drawback of detection of occult blood on a dried faecal smear is that it is not specific for gastrointestinal cancer
(especially colorectal cancer) and its sensitivity is poor. Thus many false positives are possible (with the associated drawbacks of patient anxiety, and consequent exposure to investigation such as colonoscopy, which carries significant mortality) , as well as false negatives, where patients with carcinoma are not correctly diagnosed and not subject to further investigation or treatment.
In countries such as the USA, wealthy sections of the population may choose screening by frequent colonoscopy, but this is both expensive and risky.
Examination of a fresh stool sample is an alternative technique to the above, practised on individuals known to have, or be at risk of cancer. Generally in a stool, around 90% of the DNA is bacterial. If however, human and tumour DNA can be obtained from the stool then analysis of this DNA enables screening for mutations, epigenetic alterations and markers specific for colorectal cancer.
Traditionally DNA has been obtained from stool by the patient collecting an entire stool and within a very short period of time forwarding the stool, by courier or other immediate method, to a laboratory where DNA extraction is carried out. However, as this technique requires large quantities of fresh stool sample to allow screening for DNA mutations, the examination of fresh stool material is impractical for large scale population screening
programmes. There is also the disadvantage that the sample has to be immediately forwarded to a laboratory for analysis. Thus examination of stool samples for DNA mutations in this way is only therefore currently applicable to high risk individuals and is not used on a large scale basis.
In view of the impracticality of the above techniques, large scale screening is therefore performed based on occult blood detection. This technique however lacks both sensitivity and specificity.
The limitations of the current screening techniques dictate that an improved test with improved sensitivity and specificity and which can also be used in large scale population screening programmes is desirable.
It is therefore an object of the present invention to provide a method of extracting DNA from faeces for use in the detection of disease and in particular colorectal cancer.
Accordingly, the present invention provides a method of extracting DNA from faeces comprising; smearing a small amount of the faeces onto a sample retaining support, allowing the smeared sample to dry on the retaining support, storing the sample on the sample retaining support, and extracting DNA from the sample using a standard DNA extraction procedure.
Preferably the sample retaining support is cardboard or paper.
Further preferably the sample retaining support is guaiac-impregnated card.
Alternatively the sample retaining support is nylon.
The present invention further provides a method of extracting DNA from dried faeces such that PCR can be used to determine the presence of genetic mutations / markers associated with specific diseases or conditions, comprising;
1) obtaining a stool sample,
2) smearing an amount of said stool sample onto a sample retaining support,
3) drying the smeared stool sample on the sample retaining support,
4) storing the sample for an indefinite period of time,
5) extracting the DNA from the smeared stool sample,
6) analysing the extracted DNA by means of PCR for epigenetic alterations, mutations or other markers specific for colorectal cancer.
Typically the present method will be used in the detection of gastrointestinal cancer, and in particular of colorectal cancer.
A further aspect of the present invention provides a method of extracting from a faecal sample obtained from an animal, for analysis of the presence of genetic markers specific for disease, comprising the steps of, smearing a small amount of faecal sample onto a sample retaining support, drying the sample, extracting DNA from the sample by means of a standard DNA extraction procedure and analysing the DNA by means of PCR for the presence of genetic markers indicative of disease.
Extraction of DNA from the dried faecal sample may be by any suitable technique present in the art, wherein the DNA extraction process results in there being no interference from dietary components present in smear and there being no interference from bacterial DNA present in the sample.
Preferably DNA extraction is based on the guanidinium extraction method published by Karlinsey J. et al (Simultaneous purification of DNA and RNA from small numbers of eukayotic cells) Anal Biochem. 1989. Aug 1. 180(2) p 303-306.
Alternatively the DNA can be extracted by commercially available kits, such as the Quiagen DNA extraction kit.
Examination of the extracted DNA may be by screening for any suitable tumour or cell proliferation DNA marker .
Preferably any suitable mutation or marker specific for colorectal cancer or cell dysplasia may be chosen.
Preferably screening of the extracted DNA for suitable mutations or markers is by PCR, wherein primers specific for the amplification of the area of DNA where the mutation may occur is effected.
The present invention shows that if a faecal sample is smeared onto a suitable matrix such as card, paper or nylon the sample can be stored on this card for an indefinite period of time and still permit the extraction of DNA from the sample, which can be used in analysis.
As no degradation occurs to the cellular content of the sample during storage, subsequent DNA extraction can be effected.
The method of the present invention allows extraction of DNA of a quality comparable to other more laborious, u'npleasant and expensive methods and thus is particularly advantageous due to its economy of use, convenience and further due to the stability of the sample when smeared on the card in that it exhibits long term stability even at room
temperature i.e. no refrigeration or specific sample storage conditions are required.
Further, the method of sample capture required for the present invention has several advantages over techniques known previously;
(i) subsequent screening can be carried out on a much smaller amount of faecal sample,
(ii) the patient from whom the sample is being taken does not have to collect and dispatch by courier the stool to a laboratory, where screening must be carried out on the sample immediately,
(iii)the card onto which the smear is placed can simply be forwarded to the laboratory by post,
(iv) analysis of a sample collected as described by the present invention is not so time dependant, as for the examination of fresh stool samples,
(v) sample collection is a much simplified and pleasant procedure for the patient, especially to those who may be considered to be in a high risk cancer group and thus require frequent screening.
An embodiment of the present invention will now be described by way of example only.
A small sample of faeces is placed onto a guaiac- impregnated card. The faecal sample is allowed to dry out and is then posted or taken to a laboratory for analysis.
The first step of the analytical procedure is the extraction of the DNA from the sample using the guanidinium based technique; the area of card smeared with the faecal sample is shredded and then rehydrated in guanidinium isothocyanate buffer. After incubation for rehydration, the sample is vortexed to disrupt the card. Further to spinning out the pellet debris, the supernatant is extracted several times with glass milk solution to bind the DNA. DNA is released into solution from the silica matrix by treatment with a low-salt buffer. The DNA sample is suitable for analysis by PCR.
An alternative method of extracting DNA from the dried faecal sample would comprise the steps of shredding the area of card smeared with the faecal sample and rehydrating this in guanidinium isothocyanate buffer, as in the first method. The liquid generated is then treated with proteinase K. This mixture is then extracted twice with phenol/chloroform, and finally once with chloroform. The resulting supernatant is then treated with ice cold absolute alcohol to precipitate the DNA, which can be amplified by PCR for analysis.
Alternatively DNA can be extracted using commercially available kits such as the Qiagen DNA extraction kit .
Upon extraction of the DNA from the sample, the DNA can subsequently be analysed by PCR.
Analysis by PCR would involve primers which can be used to amplify and thus show the presence of DNA normal to human or alternatively DNA analogous to tumours.
The present technique therefore allows for the sensitive and accurate extraction and analysis of DNA from faecal samples, wherein the sample can be simply and hygienically transported by the postal system to a laboratory and has the following advantages over tests previously known in the art:
(i) Improved sensitivity and specificity of the technique over current methodologies for screening for asymptomatic colorectal cancer by occult blood testing of dried faecal smears.
(ii) The technique would alleviate the difficulties involved in collecting and transporting the number of fresh stool samples required for large scale population screening via the postal system.
(iii)The technique would provide longer, cheaper and stable storage of samples.
Extraction of DNA from dried faecal smears has the advantage that tumour or cell proliferation DNA markers may be studied. In particular markers specific for colorectal cancer, cell dysplasia or neoplasia may be chosen. In contrast to occult blood screening, the present invention does not suffer from interference from dietary components and further specificity is greatly improved.
In carrying out the present invention, the following points should be noted:
(i) Human-specific DNA can be identified from the card,
(ii) DNA specific for the presence of neoplasia / cancer can be identified from patient samples, (iii)DNA markers or mutations detected in the card should correlate with patient tumour or polyp samples,
(iv) No tumour specific DNA markers should be amplified in the absence of disease.
There is extensive literature on the detection of tumour mutations from large volumes of fresh stool. Many of these papers describe the extraction of DNA from fresh stool samples. However they make no mention or suggestion of DNA extraction from occult blood cards or dried faecal smears.
The method of DNA extraction of the present invention could also have application in the veterinary field, where faecal samples from animals could be collected in the same way with the DNA being extracted and analysed for the presence of genetic markers specific for disease.
Claims
1. A method of extracting DNA from faeces comprising the steps of smearing a small sample of faeces onto a sample retaining support, allowing the sample to dry on the retaining support, extracting DNA from said sample by means of a standard DNA extraction procedure, wherein the dried sample is capable of being stored and transported on the retaining support.
2. A method as claimed in claim 1 wherein the sample retaining support is cardboard or paper.
3. A method as claimed in claim 1 wherein the sample retaining support is nylon or guaiac- impregnated.
4. A method of screening for the presence of colorectal cancer comprising the steps of smearing a small sample of faeces onto a sample retaining support, drying said sample on the support, extracting DNA from said sample and analysing the DNA by means of PCR to screen for epigenetic alterations, mutations or other markers specific for colorectal cancer.
5. The use of cardboard as a sample retaining support for the stable storage of a dried faecal sample prior to the extraction and screening of the DNA in the sample for diagnosis of colorectal cancer.
6. The use of paper as a sample retaining support - for stable storage of a faecal sample, prior to - the extraction and screening of DNA contained within the sample to allow the detection of the presence of colorectal cancer.
7. A method of extracting DNA from a faecal sample to check for the presence of genetic markers specific for disease, comprising the steps of; smearing a small amount of faecal sample onto a sample retaining support, drying the sample, extracting DNA from said sample by means of a standard DNA extraction procedure, analysing the DNA for the presence of genetic markers indicative of disease, where the sample can be stored on the support for an indefinite period of time prior to DNA extraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2001266212A AU2001266212A1 (en) | 2000-06-28 | 2001-06-28 | Method for dna extraction |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB0015777.6A GB0015777D0 (en) | 2000-06-28 | 2000-06-28 | DNA Extraction |
| GB0015777.6 | 2000-06-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2002000930A2 true WO2002000930A2 (en) | 2002-01-03 |
| WO2002000930A3 WO2002000930A3 (en) | 2002-10-17 |
Family
ID=9894538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB2001/002909 WO2002000930A2 (en) | 2000-06-28 | 2001-06-28 | Method for dna extraction |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU2001266212A1 (en) |
| GB (1) | GB0015777D0 (en) |
| WO (1) | WO2002000930A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003060156A2 (en) * | 2001-12-24 | 2003-07-24 | Tayside University Hospitals Nhs Trust | Dna detection using primers hybridizing to non-fragmentated dna |
| EP1451317A1 (en) * | 2001-11-05 | 2004-09-01 | Riken | Methods of storing and/or delivering an oligomer and/or polymer applied on a support, and supports thereof |
| EP1669737A2 (en) * | 2004-12-10 | 2006-06-14 | IPUS Industrie-Produktions- und umwelttechnisches Service GmbH | Method for determining eutrophic systems |
| WO2012004619A1 (en) | 2010-07-07 | 2012-01-12 | Diagon Kft. | Procedure for the specific isolation of total dna content of bacterial germs and a kit for this purpose |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997007239A1 (en) * | 1995-08-16 | 1997-02-27 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin | Process for purifying, stabilising or isolating nucleic acids from biological materials |
| US5939259A (en) * | 1997-04-09 | 1999-08-17 | Schleicher & Schuell, Inc. | Methods and devices for collecting and storing clinical samples for genetic analysis |
-
2000
- 2000-06-28 GB GBGB0015777.6A patent/GB0015777D0/en not_active Ceased
-
2001
- 2001-06-28 AU AU2001266212A patent/AU2001266212A1/en not_active Abandoned
- 2001-06-28 WO PCT/GB2001/002909 patent/WO2002000930A2/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997007239A1 (en) * | 1995-08-16 | 1997-02-27 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin | Process for purifying, stabilising or isolating nucleic acids from biological materials |
| US5939259A (en) * | 1997-04-09 | 1999-08-17 | Schleicher & Schuell, Inc. | Methods and devices for collecting and storing clinical samples for genetic analysis |
Non-Patent Citations (3)
| Title |
|---|
| DATABASE MEDLINE [Online] June 1996 (1996-06) RUSSOMANDO G ET AL: "Trypanosoma cruzi: polymerase chain reaction-based detection in dried feces of Triatoma infestans." Database accession no. NLM8654552 XP002203422 & EXPERIMENTAL PARASITOLOGY. UNITED STATES JUN 1996, vol. 83, no. 1, June 1996 (1996-06), pages 62-66, ISSN: 0014-4894 * |
| DATABASE MEDLINE [Online] May 2000 (2000-05) CARNEVALE S ET AL: "Diagnosis of Enterocytozoon bieneusi by PCR in stool samples eluted from filter paper disks." Database accession no. NLM10799469 XP002203421 & CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY. UNITED STATES MAY 2000, vol. 7, no. 3, May 2000 (2000-05), pages 504-506, ISSN: 1071-412X * |
| SAULNIER P ET AL: "Detection of genes in feces by booster polymerase chain reaction." JOURNAL OF CLINICAL MICROBIOLOGY. UNITED STATES AUG 1992, vol. 30, no. 8, August 1992 (1992-08), pages 2080-2083, XP002203943 ISSN: 0095-1137 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1451317A1 (en) * | 2001-11-05 | 2004-09-01 | Riken | Methods of storing and/or delivering an oligomer and/or polymer applied on a support, and supports thereof |
| EP1451317A4 (en) * | 2001-11-05 | 2005-05-11 | Riken | Methods of storing and/or delivering an oligomer and/or polymer applied on a support, and supports thereof |
| WO2003060156A2 (en) * | 2001-12-24 | 2003-07-24 | Tayside University Hospitals Nhs Trust | Dna detection using primers hybridizing to non-fragmentated dna |
| WO2003060156A3 (en) * | 2001-12-24 | 2003-11-06 | Tayside University Hospitals N | Dna detection using primers hybridizing to non-fragmentated dna |
| EP1669737A2 (en) * | 2004-12-10 | 2006-06-14 | IPUS Industrie-Produktions- und umwelttechnisches Service GmbH | Method for determining eutrophic systems |
| EP1669737A3 (en) * | 2004-12-10 | 2006-06-21 | IPUS Industrie-Produktions- und umwelttechnisches Service GmbH | Method for determining eutrophic systems |
| WO2012004619A1 (en) | 2010-07-07 | 2012-01-12 | Diagon Kft. | Procedure for the specific isolation of total dna content of bacterial germs and a kit for this purpose |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2001266212A1 (en) | 2002-01-08 |
| WO2002000930A3 (en) | 2002-10-17 |
| GB0015777D0 (en) | 2000-08-16 |
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