METHOD FOR THE DETERMINATION OF ALTERATIONS IN THE SEQUENCE OF DNA MOLECULES USING MULTIPLE-DYE CFLP (MD-CFLP)

Abstract: A method for the determination of alterations of DNA sequences using the endoclease Cleavase I and internal labelling of DNA fragments.

Description: "METHOD FOR THE DETERMINATION OF ALTERATIONS IN THE SEQUENCE OF DNA MOLECULES USING MULTIPLE-DYE CFLP (MD-CFLP)" 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. For example, in hereditary breast cancer syndrome, 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. For these reasons, extremely sensitive pre-screening methods are normally used such as SSCP. DGGE, and PTT (Friedman. L.S. et al.. Nature Genet 8, 399-404 ( 1994); Stoppa-Lyonnet, D. et al.. Am. J. Hum. Genet. 60. 1021 - 1030 ( 1997); Hogervorst. F.B.L. et al.. Nat. Gena 10. 208- 212 ( 1995). 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. Up to now, 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. In Brow M.A.D. et al, J Clin Microbiol 34, 3129-3137 (1996), the CFLP is described using automated sequencer, with terminal labelling of the fragments obtained through binding of a fluorescent group to 5 'ends of the primers. In Eisinger F. et al., Cancer Res 58, 1588-1593 (1998), a CFLP method is described applied to the analysis of exons 1 1 and 20 of BRCA1 , in which the terminal labelling of 3 ' primers is adopted. In Rossetti S. et al., Mol Cell Probes 11, 155-160 (1997), the analysis of various mutations is compared using the SSCA technique and CFLP with visualisation in silver staining. In Maddox L. O. et al, Biochem Mol Biol Int 43.1163-1171 (1997), the determination of 6 mutations on exon VII of the IDS gene is compared using the SSCP technique and CFLP with chemiluminescent detection. It has now been found that it is possible to determine several alterations of a DNA sequence with a method based on the use of the endonuclease Cleavase I and of the internal labelling of the sequence. 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. In 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. In particular, as reported in Brow M.A.D. et al., J Clin Microbiol 34, 3129-3137 (1996), e in Rosetti, S. et al., Mol. Cell Probes 11 , 155- 160 (1997), 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. In particular, internal labelling of the fragments increases the sensitivity of the method. permitting the visualisation of all the products of the reaction. Furthermore, 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. Among the possible applications, there can be cited the analysis of 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. As an example of how the invention works, the BRCA1 gene of high risk patients was analysed, verifying the presence of alterations of the sequence previously characterised and reported in Table 1. BRCA1 exons 1 1 and 16 were examined, which together cover 70% of the codifying region. 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. The samples to compare were always labelled with the same fluorocrome to avoid variations in the DNA structure due to the different dUTPs used. Once optimal conditions for CFLP were established. 18 samples were analysed with 12 different known alterations (reported in Table 1). It was observed that each sample with a specific variation in the sequence generated a single reproducible fingerprint that was easily distinguishable from the wild type and often correlated with an alteration identified before sequencing. Some typical electropherograms are reported in Figures 1 and 2, and show the exon 1 1B pattern of a patient with three common polymorphisms, and the exon 1 1 C pattern of a patient with a base substitution of an intron region. These electropherograms demonstrate a clear difference between wild type samples and altered samples, and show how the method is capable of detemining alterations near the primer used for PCR amplification, which permits the reduction of the overlapping normally required, and thus the size of the amplicon. Table 1 : Mutations and common polymorphisms of BRCAl analysed by MD- CFLP All the alterations were previously described in Breast Cancer Information Core Database (BIC) apart from the last one, a base substitution in the intronic region. Abbreviations as reported in BIC (available on Internet at the following website: www.nhgri.nih.gov/Intramural_researc/Lab_transfer/BicV l UV: non classified variant; P = polymorfismo; F = "frameshift mutation. Details of the experimental results are reported in the following example. 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 800μg of purified DNA are obtained from a 10 ml blood sample. For each PCR reaction, 100 ng of DNA are amplified in a final volume of 50 μl 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 μM [R110] or [R6G] dUTP, or 2 μM [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 μl 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 μl (20 μg) of glycogen (Boehπnger 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. Mutation Analysis 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 μl. 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 μl of reaction mix ( 0.25 mM MnCi2, 1 x reaction buffer, 1 μl (25 U) Cleavase I enzyme (Third Wave Technologies) pre-heated to the optimal temperature is then added. After 15 minutes the reaction is stopped adding 10 μl of Stop solution (95 % deionized formamide, 10 mM EDTA e 0.05% blue dextran - SIGMA). The digestion pattern is resolved by electrophoresis on 5% denaturing gel, acrylamide 19: 1 , 7 M Urea in TBE 1 x, in an ABI373A apparatus at 30 W constant power for 12 hours. By constructing the appropriate matrices, it is possible to analyse simultaneously the four BRCAl fragments labelled with the three different fluorocromes available commercially, simply by loading them into the same well at staggered times. The following loading protocol is considered satisfactory (4 μl each sample): exon 16 → run of 25 minutes → exon 1 1 C → run of 10 minutes → exon 11 B → run of 10 minutes → exon 1 1 A. Analysis is performed using GeneScan PCR Analysis Software 672 version 1.2.0 (Applied Biosystem). 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. To construct a personalized matrix, 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. Such a matrix (attached) has been verified several times, comparing the pattern of the same fragment loaded both singly and multiply. As can be seen in the matrix, there is a fourth colour, red, reserved uniquely for molecular weight standards and not used by us in this analysis; when preparing the matrix for CFLP analysis of BRCAl , therefore given a zero value. Matrix for CFLP analyses of BRCAl . Table 3 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. Analysis was carried out using 373 DNA Sequencer Data Analysis Program software (versione 1.2.0). BRIEF DESCRIPTION OF THE FIGURES 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. BIBLIOGRAPHIC REFERENCES Friedman, L.S. et al., Confirmation of BRCAl by analysis of germline mutations linked to breast =and ovarian cancer in ten families. Nature Genet 8, 399-404 (1994); Stoppa-Lyonnet, D. et al., BRCAl sequence variations in 160 individuals referred to a breast/ovarian family cancer clinic, Am. J. Hum. Genet. 60, 1021-1030 (1997); Hogervorst, F.B.L. et al., Rapid detection of BRCAl mutations by the protein truncation test, Nat. Genet 10, 208-212 (1995); Rosetti, S. Englisch, S., bresin, E., Pignattu, P.F. & Turco, A.E„ Detection of mutations in human genes by a new rapid method: cleavage =fragment length polymorphism analysis (CFLPA), Mol. Cell Probes 11, 155-160 (1997); Brow, M.A.D. et al., Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tubercolosis katG by =structure- specific endonuclease cleavage, J. Clin. Microbiol. 34, 3129-3137 (1996);

Claims: CLAIMS 1. Method for the determination of alterations of DNA sequences which comprises: a) providing a separate PCR amplification of the regions of the target DNA sequence in a reaction mixture that comprises specific primers, a thermostable polymerase, the triphosphate deoxynucelosides necessary for DNA synthesis and at least one triphosphate deoxynuceloside labelled with a fluorocrome; b) digesting the amplicons with the endonuclease Cleavase I to obtain a mix of fragments; c) separating the mix of fragments using electrophoresis and visualize the digestion pattern through band analysis. 2. Method according to claim 1, in which the target DNA is the BRCAl or BRCA2 gene. 3. Method according to claim 2, in which the target DNA is the BRCAl gene and the amplified regions correspond to exon 16 and exons 11 A, 11B and 11C, wherein 11A, 11B and 11C are the intervals 33793- 35191, 35065-36285 and 36213-37315 of the sequence GenBank accession number U 14680, respectively. 4. Method according to claim 3, wherein the labelled deoxynucleoside triphosphate is dUTP. 5. Method according to claims 1-4, wherein the electrophoretic separation is peformed on denaturing acrylamide gel using an automated sequencer capable of exciting the fluorocromes with a laser light and of detecting the fluorescence emitted by them. 6. Use of DNA fragments internally labelled with fluorocromes in a method for the determination of alterations of a target DNA based on the endonucleasic activity of the enzyme Cleavase I. 7. Use according to claim 6, wherein the target DNA is the BRCAl gene.

Inventors: CALISTRI DANIELE, CORTESI LAURA, CALISTRI, DANIELE, CORTESI, LAURA
Applicants: CALISTRI DANIELE, CORTESI LAURA, ISTITUTO ONCOLOGICO ROMAGNOLO, CALISTRI, DANIELE, CORTESI, LAURA, ISTITUTO ONCOLOGICO ROMAGNOLO COOPERATIVA SOCIALE
Application year: 2000
Main classification: C12Q1
IPC codes: C12Q1