WO2000055360A2 - Method for the determination of alterations in the sequence of dna molecules using multiple-dye cflp (md-cflp) - Google Patents
Method for the determination of alterations in the sequence of dna molecules using multiple-dye cflp (md-cflp) Download PDFInfo
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- WO2000055360A2 WO2000055360A2 PCT/EP2000/002054 EP0002054W WO0055360A2 WO 2000055360 A2 WO2000055360 A2 WO 2000055360A2 EP 0002054 W EP0002054 W EP 0002054W WO 0055360 A2 WO0055360 A2 WO 0055360A2
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- dna
- cflp
- alterations
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- sequence
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention provides a method for the determination of alterations of the polynucleotide sequence of DNA molecules, such as mutations, deletions, insertions, substitutions, or, in general, variations in the nucleotide sequence.
- the identification of the alterations present in DNA sequences, especially in gene sequences, is extremely important for the diagnosis of genetic diseases or in the determination of a predisposition to develop pathologies associated with a particular/specific genetic anomaly.
- two main genes known as BRCA1 e BRCA2 have been identified as being involved in the breast cancer predisposition. More than 200 mutations scattered along the entire length of these two genes have been identified.
- the complete sequencing of the genes permits an accurate identification of the mutations but is an extremely laborious, costly and time- consuming procedure, especially if we take into consideration the size of the genes involved.
- extremely sensitive pre-screening methods are normally used such as SSCP. DGGE, and PTT (Friedman. L.S.
- the CFLP method (“Cleavase Fragment Length Polymorphism”. CFLP- Third Wave Technologies) was examined in an attempt to improve the sensitivity and efficacy of the pre-sequencing screening procedure. This method is capable of determining single or multiple base alterations in fragments of up to 2kb in length.
- the CFLP analysis uses a thermostable structure-specific endonuclease. Cleavase I. which specifically recognizes and cleaves near the 5' ends of "hairpins", secondary structures that generate intramolecularly within single-strand DNA during DNA renaturation.
- the formation of the secondary structure is closely correlated with the DNA sequence under investigation and with the temperature; it follows that at the predetermined temperature, digestion with Cleavase I generates a specific series of fragments. The products of this digestion are separated using electrophoresis on denatured polyacrylamide gel, obtaining a distribution of bands which makes up a distinctive imprint, specific for the sequence analysed. Mutations such as base substitutions, insertions or deletions influence the formation of secondary structures and thus the electrophoretic distribution in which the appearance or disappearance of one or more bands and the increase or reduction in the intensity of the signal with respect to the "wild type" fingerprint is observed.
- the CFLP method has been used in the differentiation of microbial species, in the genotyping of the hepatitis C virus, and has been indicated as an alternative to SSCP, HA and DGGE methods for the analysis of large-scale mutations.
- the method of the invention comprises the following steps: a) providing a separate PCR (Polymerase Chain Reaction) amplification of the regions of the target DNA sequence in a reaction mixture that includes the specific primers, a thermostable polymerase, triphosphate deoxynucleosides needed for DNA synthesis, and at least one triphosphate deoxynucleoside labelled with a fluorocrome; b) Digesting the amplicons with endonuclease Cleavase I in order to obtain a mixture of fragments; c) Separating electrophoretically the mixture of fragments and visualising the digestion pattern through band analysis.
- the fluorocromes with which the triphosphate deoxynucleosides are labelled can be selected from those available commercially under the names TAMRA, R6G, Rl 10. It is preferable to conduct the labelling on dUTP.
- step a regions of dimensions between 300 and 2000 base pairs (bp) can be amplified, preferably between 400 and 1500 bp.
- the amplicons that undergo digestion with Cleavase I generate fragments of different lengths which are distinguishable by means of internal labelling with fluorocrome.
- the system permits the contemporary analysis (in the same well) of more amplicons.
- the maximum number of amplicons that can be analysed simultaneously depends on the fluorocromes available and on the relative dimensions of the fragments generated starting from the amplicons themselves. in particular, from the possibility of distinguishing fragments on the basis of their relative electrophorectic migration or of the fluorocrome with which they are labelled.
- the general conditions for the digestion with Cleavase I can be optimized each time, trying to avoid overdigestion of the sample and ensuring the reproducibility of the distribution pattern of the fragments.
- the digestion temperature is an important variable for obtaining good sensitivity for each sample, whereas the saline concentration, in particular the concentration of MnCl2 needed for the enzymatic activity, and the reaction time, can easily be determined.
- the fragments obtained from digestion with Cleavase I are separated on acrylamide denaturing gel using an automatic sequencer capable of exciting the fluorocromes by laser light and of displaying the fluorescence emitted by them, permitting a visualisation of the bands corresponding to the different fragments and their relative intensity.
- the automatic sequencer of choice is the AB 1373A model provided by Applied Biosystem, and the electrophoretic scanning is analysed using GeneScan software. Normally, the visualisation pattern of the sample under investigation is compared with that of a control sample in order to highlight any difference connected with alterations in the DNA sequence of the test sample with respect to the control sample. Such differences may correspond to deletions, insertions, substitutions or any sequence variation which involves a change in the secondary structure of the amplicons that is recognised by the enzyme Cleavase I.
- the invention method offers a series of advantages with respect to techniques commonly used in the preliminary screening of DNA sequences for the identification of any alterations in the target sequences.
- internal labelling of the fragments increases the sensitivity of the method. permitting the visualisation of all the products of the reaction.
- it is possible to analyse simultaneously both nucleic acid chains without having to resort to separate reactions, and this is particularly important in consideration of the fact that some alterations influence the CFLP pattern of a chain to a greater or lesser extent than a complementary chain (Brow, M.A.D. et al., J. Clin. Microbiol. 34, 3129-3137, 1996). Equally as important, the method is quick and easy to set up and has contained costs for reagents and equipment.
- nucleotide variations, mutations or polymorphisms in known sequences starting from both genomic DNA and cDNA; for example, analysis of germinal or somatic mutations in genes involved in tumor transformation or in the onset of genetic diseases, fine characterisation of microorganisms, study of polymorfisms and allelic frequences.
- the BRCA1 gene of high risk patients was analysed, verifying the presence of alterations of the sequence previously characterised and reported in Table 1.
- Exon 1 1 which extends for more than half of the gene (3426 bp), was subdivided into 3 partially overlapping regions [GenBank accession number U14680: HA(nt 33793-35191), 1 1B (nt 35065 - 36285) and 1 1C(36213 - 37315)], including the flanking regions of the introns. These three amplicons and exon 16 (with flanking sequences of the introns) were labelled through incorporation of different fluorescent dUTPs during amplification for PCR.
- EXAMPLE Method of mutation analysis of BRCAl a) DNA extraction and amplification with the Polymerase Chain Reaction (PCR).
- the DNA is extracted from peripheral blood lymphoctyes following the classic method of extraction with phenol-chloroform. From 400 to 800 ⁇ g of purified DNA are obtained from a 10 ml blood sample.
- PCR reaction 100 ng of DNA are amplified in a final volume of 50 ⁇ l containing 10 pmol of primer (described in Table 2 below), 0.2 mM of each dNTP, 1-1.5 mM MgCl2 according to the specific reaction, 2 units of Taq-polymerase, 1 X reaction buffer and 0.5 ⁇ M [R110] or [R6G] dUTP, or 2 ⁇ M [TAMRA] dUTP (Applied Biosystem).
- primer described in Table 2 below
- 0.2 mM of each dNTP 1-1.5 mM MgCl2 according to the specific reaction
- 2 units of Taq-polymerase 1 X reaction buffer and 0.5 ⁇ M [R110] or [R6G] dUTP, or 2 ⁇ M [TAMRA] dUTP (Applied Biosystem).
- PCR amplification is thus complete: after initial denaturation at 94 °C for two minutes, 40 cycles with: denaturation at 94 °C for one minute, annealing of the primers at 58 °C for one minute and 30 seconds, extension at 72 °C for one minute. The last step is 5 minutes at 72 °C.
- PCR product are precipitated in ammonium acetate 2 M and two volumes of isopropanol for 10 minutes at room temperature, centrifuged for 20 minutes at 13000 rpm and the pellet is washed in Ethanol 70%; after drying, the pellet is resuspended in 20 ⁇ l of sterile distilled water.
- This step eliminates non- incorporated dUTPs and primers, and removes reaction salts that interfere with the digestion of Cleavase I and with the re-annealing of the denatured DNA.
- Precipitation yield increases by adding 1 ⁇ l (20 ⁇ g) of glycogen (Boeh ⁇ nger Mannheim).
- the concentration of PCR products is estimated using electrophoresis on agarose gel containing ethidium bromide.
- Table 2 BRCA l primers and reaction conditions of CFLP.
- PCR products For each CFLP reaction, 200 - 400 ng of PCR products (labelled with fluorescent dUTPs) are diluted in sterile distilled water, with a final reaction volume of 20 ⁇ l. Samples are denatured at 94 °C for 30 seconds and then rapidly brought to an optimal predetermined temperature for each fragment (reported in Table 2), on which form secondary structures of single DNA strands. 5.5 ⁇ l of reaction mix ( 0.25 mM MnCi2, 1 x reaction buffer, 1 ⁇ l (25 U) Cleavase I enzyme (Third Wave Technologies) pre-heated to the optimal temperature is then added.
- reaction mix 0.25 mM MnCi2, 1 x reaction buffer, 1 ⁇ l (25 U) Cleavase I enzyme (Third Wave Technologies) pre-heated to the optimal temperature is then added.
- Personalized matrices can be constructed with this software that are capable of accurately attributing signal intensity in fluorescence to a specific colour and thus fragment, subtracting the background noise linked to the contemporary presence of other fluorocromes.
- Personalized matrices can be constructed with this software that are capable of accurately attributing signal intensity in fluorescence to a specific colour and thus fragment, subtracting the background noise linked to the contemporary presence of other fluorocromes.
- different fragments amplified and labelled with several fluorocromes are loaded both singly and multiply. Using the fragments loaded singly as reference standards, the ratios between the different fluorocromes are modified in such a way that, from analysis of the multiple loading, the same result is reproduced, that is, the digestion pattern with Cleavase I typical of the nucleotide sequence in question.
- Nucleotide sequence analysis Each sample with a suspect pattern from the CFLP analysis is re- analysed and, if confirmed, sequenced with internal primers to amplified fragments using the PRISM Dye Terminator Cycle Sequencing kit for the 373A automated sequencer (Applied Biosystem). The sequence is resolved on 6% denaturing gel Acrilamide 19: 1 , 7 M Urea, 1 x TBE at 30 W constant power for 12 hours.
- FIGURE 1 Electropherograms of exon 11B; comparison between a sample with three common polymorphisms and the wild type sample. The main differences are indicated with arrows. The same altered electropherogram was obtained for all the 11 samples bearing the same polymorphisms.
- FIGURE 2 Electropherograms of exonl lC: comparison between a sample bearing a base substitution in the intronic region and a wild type sample. The main differences are indicated with arrows.
- cleavage fragment length polymorphism analysis (CFLPA), Mol. Cell Probes 11, 155-160 (1997);
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00918777A EP1159456A2 (en) | 1999-03-12 | 2000-03-09 | Method for the determination of alterations in the sequence of dna molecules using multiple-dye cflp (md-cflp) |
AU39615/00A AU3961500A (en) | 1999-03-12 | 2000-03-09 | Method for the determination of alterations in the sequence of dna molecules using multiple-dye cflp (md-cflp) |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITMI99A000512 | 1999-03-12 | ||
IT1999MI000512A IT1311896B1 (en) | 1999-03-12 | 1999-03-12 | METHOD FOR DETERMINING SEQUENCE ALTERATIONS OF MOLECOLEDI DNA BY MULTIPLE-DYE CFLP (MD-CFLP). |
Publications (2)
Publication Number | Publication Date |
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WO2000055360A2 true WO2000055360A2 (en) | 2000-09-21 |
WO2000055360A3 WO2000055360A3 (en) | 2000-12-28 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/EP2000/002054 WO2000055360A2 (en) | 1999-03-12 | 2000-03-09 | Method for the determination of alterations in the sequence of dna molecules using multiple-dye cflp (md-cflp) |
Country Status (4)
Country | Link |
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EP (1) | EP1159456A2 (en) |
AU (1) | AU3961500A (en) |
IT (1) | IT1311896B1 (en) |
WO (1) | WO2000055360A2 (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0657548A1 (en) * | 1993-12-10 | 1995-06-14 | Becton, Dickinson and Company | Amplification of nucleic acids in cells |
WO1997043441A1 (en) * | 1996-05-14 | 1997-11-20 | Visible Genetics Inc. | Method and reagents for testing for mutations in the brca1 gene |
WO1998050403A1 (en) * | 1997-05-05 | 1998-11-12 | Third Wave Technologies,Inc. | Target-dependent reactions using structure-bridging oligonucleotides |
US5843654A (en) * | 1992-12-07 | 1998-12-01 | Third Wave Technologies, Inc. | Rapid detection of mutations in the p53 gene |
-
1999
- 1999-03-12 IT IT1999MI000512A patent/IT1311896B1/en active
-
2000
- 2000-03-09 WO PCT/EP2000/002054 patent/WO2000055360A2/en not_active Application Discontinuation
- 2000-03-09 AU AU39615/00A patent/AU3961500A/en not_active Abandoned
- 2000-03-09 EP EP00918777A patent/EP1159456A2/en not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5843654A (en) * | 1992-12-07 | 1998-12-01 | Third Wave Technologies, Inc. | Rapid detection of mutations in the p53 gene |
EP0657548A1 (en) * | 1993-12-10 | 1995-06-14 | Becton, Dickinson and Company | Amplification of nucleic acids in cells |
WO1997043441A1 (en) * | 1996-05-14 | 1997-11-20 | Visible Genetics Inc. | Method and reagents for testing for mutations in the brca1 gene |
WO1998050403A1 (en) * | 1997-05-05 | 1998-11-12 | Third Wave Technologies,Inc. | Target-dependent reactions using structure-bridging oligonucleotides |
Non-Patent Citations (2)
Title |
---|
EISENGER F ET AL: "Mutations at BRAC1: The medullary breast carcinoma revisited" CANCER RESEARCH, vol. 58, April 1998 (1998-04), pages 1588-92, XP000946319 cited in the application * |
GANGULY T ET AL: "HIGH THROUGHPUT FLUORESCENCE-BASED CONFORMATION-SENSITIVE GEL ELECTROPHORESIS (F-CSGE) IDENTIFIES SIX UNIQUE BRCA2 MUTATIONS AND AN OVERALL LOW INCIDENCE OF BRCA2 MUTATIONS IN HIGH-RISK BRCA1- NEGATIVE BREAST CANCER FAMILIES" HUMAN GENETICS,DE,BERLIN, vol. 102, no. 5, 1998, pages 549-556, XP000874587 * |
Also Published As
Publication number | Publication date |
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EP1159456A2 (en) | 2001-12-05 |
AU3961500A (en) | 2000-10-04 |
WO2000055360A3 (en) | 2000-12-28 |
ITMI990512A1 (en) | 2000-09-12 |
IT1311896B1 (en) | 2002-03-20 |
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