MXPA00010281A - A method for the characterisation of nucleic acid molecules involving generation of extendible upstream dna fragments resulting from the cleavage of nucleic acid at an abasic site - Google Patents

Abstract

A method for characterising nucleic acid molecules comprises the steps of:i) introducing a modified base which is a substrate for a DNA glycosylase into a DNA molecule;ii) excising the modified base by means of said DNA glycosylase so as to generate an abasic site;iii) cleaving the DNA at the abasic site so as to generate an upstream DNA fragment that can be extended;and iv) incubating the extendible upstream fragment in the presence of an enzyme, for example a polymerase or a ligase, allowing for extension thereof and a template nucleic acid and analysing the resultant fragment(s). The invention provides a novel, versatile and simple method using the above-mentioned extendible upstream DNA fragments which allows characterisation of nucleic acids and which has advantages over existing methods. One of the most important uses (but not the only use) of the method according to the invention is to scan or check a fragment of DNA (target nucleic acid) for the presence or absence of a mutation.

Description

A METHOD FOR THE CHARACTERIZATION OF ACID MOLECULES NUCLEUS THAT INCLUDES THE GENERATION OF DNA FRAGMENTS WHICH CAN BE EXTENDED IN DIRECTION 5 'RESULTING FROM THE NUCLEIC ACID EXCISION IN AN ABASIC SITE FIELD OF THE INVENTION The present invention relates to a method for characterizing nucleic acid molecules, which involves the generation of DNA fragments that can extend in the 5 'direction, which result from the cleavage of the nucleic acid at an abasic site.

BACKGROUND OF THE INVENTION The characterization of target nucleic acids is very important for several reasons that are related to the confirmation of the presence or absence of a gene in a sample, of the confirmation of part or all of a nucleic acid sequence and the detection early presence of a known or unknown disease that causes mutations that lead to inherited diseases and natural variations in DNA. Although there are many known methods for the characterization of nucleic acids and for the detection of unknown sequence changes, the increase in the amount of new genetic information that is generated, highlights the importance of developing new, better and faster methods for the characterization of the nucleic acids. WO 97/03210 discloses the use of a DNA-glycosylase enzyme, which recognizes a modified base, for the direct detection of known and unknown mutations in a target nucleic acid sample. The method typically includes amplifying a target nucleic acid sample, using a combination of normal DNA precursor nucleotides and one or more modified precursor nucleotides, wherein the modified precursor nucleotide replaces one of the normal precursor nucleotides, which is a substrate of a DNA-glucosilase After cleavage of the modified base by glycosylase, the resulting abasic site is cleaved and the products of the cleavage are analyzed. This method allows the detection of mutations in the candidate loci. However, the method of WO 97/03210 has certain limitations. For example, with this method it is not possible to detect sequence differences between nucleic acid molecules without detecting sequence similarities and, thus, multiple samples can not be combined for simultaneous analysis.

SUMMARY OF THE INVENTION The invention provides a method for characterizing nucleic acid molecules, comprising the steps of: i) introducing a modified base which is the substrate of a DNA glycosylase into a DNA molecule; ii) split the modified base by means of the DNA glycosylase to generate an abasic site; iii) cleaving the DNA at the abasic site to generate a DNA fragment in the 5 'direction that can be extended; and iv) incubating the extendable fragment in the direction 'in the presence of an enzyme that allows the extension thereof and a template nucleic acid and analyze the resulting fragments. The invention provides a novel, versatile and simple method using the above-mentioned DNA fragments that can be extended in the 5 'direction, which allows the characterization of the nucleic acids and which has the advantages with respect to the existing methods as indicated in next description. One of the most important uses (although not the only use) of the method according to the invention is to scan or check a DNA fragment (white nucleic acid) in search of the presence or absence of a mutation. The method consists essentially of i) the P1155 generation of the extensible DNA fragments in the 5 'direction and ii) the subsequent use of these fragments in the analysis of a piece of DNA (for example, detecting a mutation). Preferably, the modified base is used as uracil, hypoxanthine or 8-OH guanine. Preferably, the modified bases are derived from modified precursor nucleotides, which when incorporated into the DNA generate the modified bases. In this way, the preferred modified precursor nucleotides are dUTP, dITP and 8-OH dGTP, which when incorporated into the DNA generate the bases uracil, hypoxanthine and 8-OH guanine of the glycosylase substrate, respectively. Each of the modified precursor nucleotides is a sugar-phosphate base comprising the base portion and a sugar-phosphate portion. Uracil in DNA is specifically recognized by uracil-DNA glycosylase (UDG) and is released from DNA. The UDG also recognizes other bases related to uracil when it is present in the DNA. Hypoxanthine is specifically recognized by the alkylpyrin-DNA glycosylases (ADG) and is released from DNA. This enzyme also recognizes and releases N3 methyladenine, N3 methylguanine, O2 methylcytosine and O2 methyliltimine when present in DNA. 8-OH guanine is specifically recognized by formamido- P11SS pyrimidine DNA-glycosylase (FPG) and is released from DNA. This enzyme also recognizes and releases open-ring purines when they are present in DNA. Thymine-DNA glycosylase recognizes and releases uracil and thymine placed opposite to guanine bases in DNA. Modified precursor nucleotides, as used herein, refer to one or more modified nucleotides that can be incorporated into a nucleic acid so as to generate a modified base or bases that are recognized and can be cleaved by the enzyme DNA-glycosylase . After the introduction of the modified base, the DNA product is treated with a suitable DNA-glycosylase enzyme that recognizes and releases the base of the glycosylase substrate present in the target sample and, consequently, generates an apurinic or apyrimidine site (AP ), depending on the nature of the modified bases. The AP site is the term given to a site in the DNA, where the base portion of a nucleotide has been lost or deleted, leaving behind a deoxyribophosphate with the DNA phosphodiester backbone still intact. AP is the abbreviation for either apurinic and / or apyrimidine, depending on whether a purine or pyrimidine base has been attached to the ribose ring. An AP site is also referred to as an abbasic site, which is the general term for the apurinic and apirimidine site. The release of the base of the glycosylase substrate from the nucleic acid sample results in, for example, an apyrimidine site in the case of uracil and in an apurinic site in the case of hypoxanthine and 8-OH dG. Collectively, these sites are referred to as abasic sites. Essentially, the excised fragments must have hydroxyl groups at the 3 'ends and the DNA immediately in the 3' end directions must not be blocked in a way that prevents extension of the fragment from the 3 'ends. Split fragments are generated that have hydroxyl at the 3 'ends and in the 3' direction the blocking groups that prevent the fragment extension from the 3 'ends were eliminated, while being used as a template, that DNA from which it was derived the extendable fragment. The DNA can be excised in various forms at the abasic site to generate the DNA fragment in the 5 'direction as described in more detail below. For example, phosphate bonds at the abasic sites can be cleaved by a treatment selected from the treatment with a basic solution or other chemical treatment, heat treatment and / or treatment with an enzyme.

According to one embodiment of the invention, the 5 'fragment is generated by cleaving the DNA on the 5' side of the abasic site, such that the 3 'end of the 5' fragment carries a hydroxyl group. The terms "extendable fragment and DNA fragment in the 5 'direction are used interchangeably herein. Since the 3'-OH ends are generated in this manner, no further processing of the fragments is required in the 5 'direction before step iv). Treatment with basic (alkaline) solutions at high temperature or with a chemical compound such as piperidine or with an enzyme that specifically cuts at the abasic sites, such as E. coli endonuclease IV, results in excision of the abbasic site in the 5 'side. Suitably in this embodiment, excision is achieved with an 5 'endonuclease AP. According to an alternative embodiment, the fragment in the 5 'direction is generated by cleaving on the side 51 of the abasic site, so that at the end 31 of the fragment in the 5' direction there remains a phosphate group and eliminates the phosphate group, so that the 5 'fragment carries a hydroxyl group at the 3' end.

Alternatively, alkali cleavage or the abduction endonuclease activity of FPG was used, followed by removal of the 3 'phosphate. For example, the 3 'phosphate groups can be removed by enzymatic means, using enzymes with 3' phosphate activity, such as T4 polynucleotide kinase. According to a further embodiment, the fragment in the 5 'direction is generated by cleavage on the side 31 of the abasic site, so as to generate a deoxyribose phosphate group at the 3' end of the fragment in the 5 'direction and, subsequently, eliminate the deoxyribose group to leave a hydroxyl group at the 3 'end. This can be achieved by using an enzyme with 3 'deoxyribophosphodiesterase activity or by using FPG followed by a 3' phosphatase. In an alternative embodiment, the deoxyribose portions 51 'from the 3' end of the fragment in the 5 'direction are eliminated, so that the fragment in the 5' direction can extend into the template. Preferably, the 5 'deoxyribose portions are removed by a 5' deoxyribophosphodiesterase. Treatment only at elevated temperature or with a 3 'AP endonuclease, results in cleavage of the abbasic site until its termination on the 3' side.

P1155 Glycosylase-mediated cleavage cuts the extended primer to an abasic site subsequent to release of the modified base by DNA glycosylase that produces 3 'ends with 3'-OH or 3' phosphate groups or with deoxyribosphate groups. Except in the cases where 3 '-OH ends were generated, all other ends require additional processing before the extension of the fragment in the 5' direction. The glycosylase-mediated cleavage in the method according to the invention refers to both the 5 'and the 3' cleavage, including any subsequent treatment that is necessary to generate a 3 'OH group at the 3' end of the fragment in 5 'direction. Preferably, the modified base is introduced by enzymatic amplification of the DNA. Preferably, the DNA (hereinafter also referred to as the target nucleic acid) is amplified using normal DNA precursor nucleotides and at least one modified precursor nucleotide. The precursor nucleotides in the case of a DNA amplification process refer to the deoxyribonucleotides dATP, dCTP, dGTP and dTTP, referred to herein as "normal" DNA precursor nucleotides. The term "amplification", as used P1155 herein refers to any in vitro process to increase the number of copies of a sequence or nucleotide sequences. The amplification of a target nucleic acid molecule results in the incorporation of precursor nucleotides into the DNA to be amplified. Normally, the amplification of a blank sample is performed using appropriate primers in the polymerase chain reaction (PCR). During amplification, the primers bind to the target nucleic acid and extend using a DNA polymerase 5 'to 3' in the target nucleic acid, which acts as a template for the synthesis of a new DNA. The use of flanking primers, which are referred to herein as initial primers, which are attached to the upper and lower chains of a DNA molecule allow the exponential amplification of the DNA segment bounded by the upper and lower primers. The amplification will typically include the amplification of a target nucleic acid using a combination of normal DNA precursor nucleotides and one or more modified precursor nucleotides, wherein the modified precursor nucleotide replaces all or a proportion of one of the normal precursor nucleotides. Amplification of a nucleic acid using normal DNA precursor nucleotides results in the incorporation of the four normal bases G, A, T, or C into the DNA. Amplification of a nucleic acid using a modified precursor nucleotide in place of one of the normal precursor nucleotides results in the incorporation of a substrate base of the glycosylase into the DNA, in the place of one of the four normal bases G, A, T or C. The white nucleic acid sample will normally be DNA. However, RNA can also be used after conversion to DNA by reverse transcription. When a modified precursor nucleotide replaces a proportion of one of the normal precursor nucleotides, the ratio of the modified precursor nucleotide in the normal precursor nucleotide it is replacing is such that the optimum of a modified precursor nucleotide is incorporated by amplified DNA strand. This allows the subsequent cleavage of the amplified DNA strand into two fragments after contact with DNA glycosylase and an abbasic site cleavage agent as described herein. This method of cleavage will be referred to herein as glycosylase-mediated cleavage. A higher proportion of the modified precursor nucleotide is used to the normal precursor nucleotide to generate more than one site of P1155 chain excision of amplified DNA. Incorporation of a modified precursor nucleotide into the amplified product generates one or more bases modified at one or more positions recognized by a DNA glycosylase enzyme in the amplified product. Replacing the entire normal precursor nucleotide with a modified precursor nucleotide in the amplification reaction if used in step i) allows for glycosylase-mediated cleavage of an extended primer in an amplification reaction at the first 3 'position of the primer extended, where a normal base is replaced by a modified base. Thus, if the template sequence immediately 3 'from a location where the primer hybridizes is CTAG and the modified nucleotide precursor is dUTP that replaces dTTP, then the modified uracil (U) base will be incorporated opposite to A in the mold chain. Thus, in this situation, the primer will have spread in two nucleotides at the 3 'end (primer-GA 3') after amplification and glycosylase-mediated cleavage. These extended primers generated after the initial extension and the glycosylase-mediated cleavage are referred to herein, inter alia, as extensible fragments, as indicated above, whereas the primers before extension are referred to herein as the primers initials. The extended 3 'end sequence of the extendable fragment is synthesized enzymatically and is directly related to the nucleic acid which will be characterized as the nucleic acid acts as the template of its synthesis. In this way, the end 31 of the extensible fragment is complementary to the nucleic acid. Accordingly, the determination of the nature of the 3 'end of the extensible fragment by any means allows characterization of the nucleic acid from which it was derived. If an initial primer is placed adjacent to a place where a variation in the DNA sequence such as a polymorphism or a mutation occurs, so that the modified first base incorporated in the extended primer is at the mutation site, then the The initial primer will be extended in a different length, depending on whether a mutation is present after the glycosylase-mediated cleavage at the mutation site. The extensible fragments are subsequently treated (if necessary) so that they can be used as primers for a subsequent extension reaction. Because the sequence at the 3 'ends of the extensible fragments differ depending on whether a mutation is present or absent at the mutation site, the analysis of the ability of a P1155 extendable fragment functioning in a subsequent extension reaction using a nucleic acid template, allows the determination of whether a mutation is present or absent at the mutation site. Any template synthesized in enzymatic or chemical form or that occurs naturally, which is fully or partially hybridized to the extendable fragment can be designed and / or selected as a template nucleic acid that allows the ability of the extendable fragment to function as a primer that will be determined. When a proportion of a normal precursor nucleotide is replaced with a modified precursor nucleotide in the amplification reaction, glycosylase-mediated cleavage of the extended primer in an amplification reaction will produce a population of extensible fragments of various lengths, because different molecules will be cleaved at different points, depending on whether the modified precursor nucleotide is incorporated. The length of each fragment is determined by the position of the incorporation of the modified precursor nucleotide during extension of the 3 'end of the initial primer. Amplification of a target nucleic acid using the precursor nucleotides dATP, dCTP, dGTP, dTTP and a small amount of the modified precursor nucleotide dUTP results in an amplified DNA where thymine is randomly replaced by uracil. Uracil is incorporated into the newly synthesized DNA strand at complementary positions to the adenine residues in the template DNA chain during the amplification process. Amplification of a target nucleic acid using the precursor nucleotides dATP, dCTP, dGTP, dTTP and a small amount of the modified precursor nucleotide dITP results in an amplified DNA, where guanine is preferably replaced randomly by hypoxanthine. Hypoxanthine is incorporated into the newly synthesized DNA strand at complementary positions to the cytosine residues in the template DNA chain during the amplification process, when the other precursor nucleotides are not limiting. Amplification of a target nucleic acid using the precursor nucleotides dATP, dCTP, dGTP, dTTP and a small amount of the modified precursor nucleotide 8-OH dGTP, results in an amplified DNA, where guanine is preferably randomly replaced by 8- OH guanina. 8-OH guanine is incorporated into the newly synthesized DNA strand at complementary positions to the cytosine residues in the template DNA strand during the amplification process when the other precursor nucleotides are not limiting.

P1155 The amplified DNA strands can be separated for separate analysis of the respective strands. In addition, the separate chains can be immobilized, which can be achieved by various means. A common method for the immobilization and / or separation of DNA strands is through the use of the streptavidin biotin interaction, where normally, the DNA contains the biotin label and the streptavidin is bound to a solid support. However, the method according to the invention in its various steps is susceptible to the immobilization formats that allow immobilization of the fragment in the 5 'direction of the chain that carries the fragment in the 5' direction, of the chain complementary to the chain carrying the 5 'fragment of the template nucleic acid, the target nucleic acid and the products generated from the glycosylase-mediated cleavage of amplified or extended nucleic acids carrying modified bases. The modified base can be introduced by chemical modification of a nucleic acid, rather than by an amplification technique such as PCR. There are several methods in which the treatment of DNA with specific chemical compounds modifies the existing bases, so that these are recognized by specific enzymes of DNA glycosylase. For example, the treatment of DNA with alkylating agents, such as methylnitrosourea generates several alkylated bases including N3-methyladenine and N3-methylguanine which are recognized and cleaved by alkylpurin-DNA glycosylase. The treatment of DNA with sodium bisulfite causes the deamination of the cytosine residues in the DNA to form uracil residues, which are recognized and cleaved by uracil-DNA glycosylase. The treatment of DNA with ferrous sulfate and EDTA causes the oxidation of the guanine residues in the DNA to form 8-OH guanine residues, which are recognized and cleaved by formamido-pyrimidine DNA-glycosylase. In this way, the bases present in the nucleic acid or, of course, the 5 'extendable fragment generated in step iii) can be converted to chemically modified bases. A proportion or all of the cytosine residues can easily be converted to a uracil, using sodium bisulfite, thereby making the amplified sample susceptible to cleavage by uracil-DNA glycosylase at the cytosine conversion sites. If the upper or lower primer is synthesized to contain 5-methylcytosine instead of cytosine, in this case the primer will be resistant to uracil-DNA-glucosylase-mediated cleavage, since the deamination of 5-methylcytosine P1155 occurs at a reduced rate compared to cytosine and generates a thymine instead of a uracil residue. Prior to treatment with suitable DNA glycosylase, the double-stranded DNA can be treated with exonuclease I. This treatment serves to digest unused primers and any non-specific single-stranded DNA amplification products, thereby improving the signal-to-noise ratio. In the case where the modified precursor nucleotide is dUTP, the modified uracil base will be generated at the thymine positions in the amplified white nucleic acid sample. The addition of uracil-DNA glycosylase to the sample releases the uracil from the sample. In the case where the modified precursor nucleotide is dITP, the modified hypoxanthine base will be generated at the guanine positions of the amplified target nucleic acid sample. The addition of alkylpurine-DNA glycosylase to the sample liberates hypoxanthine from the sample. In the case where the modified precursor nucleotide is 8-OH dGTP, the modified 8-OH guanine base will be generated at the guanine positions of the amplified target nucleic acid sample. The addition of formamido-pyrimidine DNA-glycosylase to the sample releases the 8-OH guanine from the sample. Suitably a primer or one or more nucleotides involved in the enzymatic amplification P1155 is labeled or marked. The initial primer used may be appropriately labeled. The labeling of the primers can be performed by a variety of means including the addition of a radioactive, fluorescent or detectable ligand to the primer during or after the synthesis of the primer. In one embodiment of the invention, the enzyme used in step iv) is a polymerase that can be incubated with the 5 'extendable fragment in the presence of one or more nucleotides. Also in this embodiment, suitably one or more of the nucleotides of step iv) is a dideoxy nucleotide. One or more of the nucleotides of step iv) can also be labeled. Various nucleic acid polymerases can be used to extend the 3 'end of the extendable fragment into a template nucleic acid. Many polymerases are described in the literature that extend the 3 'ends of the primers in a template DNA. For example, DNA polymerases isolated from phage and mesophilic and thermophilic bacteria can be used. Several DNA polymerases, including T7 DNA polymerase, incorporate terminator nucleotides P1155 dideoxy as well as normal precursor nucleotides during the extension of the primers. Thermophilic DNA polymerases are routinely used in the amplification of nucleic acids by the repeated cyclic extension of the primers. The 5 'DNA fragments generated in step iii) function as primers for all nucleic acid polymerases capable of extending standard nucleic acid primers. The use of a labeled precursor nucleotide or a dideoxy terminator nucleotide in any of the extension reactions facilitates the detection of the extended extensible fragment. Direct DNA staining methods, such as silver staining or ethidium bromide, facilitate the detection of all extension products after size separation based on electrophoretic mobility. The ability of extendable fragments to function in a subsequent extension reaction using a template nucleic acid and normal terminator nucleotides or dideoxy (a nucleotide that prohibits the further extension of a primer in a template once it is incorporated) generates a fragment ladder which allow the determination of the location of the total number of positions of the modified precursor nucleotide in one or both chains of the amplified target nucleic acid. The P1155 presence of a variation in the sequence or a mutation results in the appearance or disappearance of a cleavage fragment as judged by comparison with the known DNA sequence of the amplified molecule. The size analysis of the fragments allows the location and precise sequence of a mutation in the target nucleic acid sample that will be determined. Thus, if a variation in the sequence is presented such that an additional site of incorporation of the modified precursor nucleotide is generated, an additional cleavage fragment will be observed with the analysis of the cleavage of cleavage products. If a variation in the sequence occurs such that a modified precursor nucleotide incorporation site is lost, the corresponding excision fragment will not be observed with the analysis of the cleavage of cleavage products. The mold of choice in this case will be the nucleic acid amplified originally intact or cleaved with glycosylase. In cases where nucleic acid amplified and cleaved with glycosylase is used, it can be processed to remove the residual portions in the 3 'direction of the extendable fragment, which prohibits the extension of the extendable fragment by means of a nucleic acid polymerase. Specifically, the template DNA can be treated so that a deoxyribose portion is removed P1155 5 'residual. This is achieved by incubating the template DNA with a 5 'deoxyribophosphodiesterase, such as E. coli RecJ endonuclease or formamido-pyrimidine DNA glycosylase. Other naturally occurring or enzymatically synthesized or chemically synthesized template nucleic acids that fully or partially hybridize the extendable fragment can also be designed and / or selected as a template nucleic acid to determine the ability of the extendable fragment to function as a primer. The ability of extensible fragments to function in a subsequent extension reaction using a template nucleic acid and a combination of labeled or unlabeled normal terminators or dideoxy nucleotides allows sequence and mutation variations to be detected. The extent of the extensible fragments in the template nucleic acid from which they were derived and which is a heterozygous of the sequence variation, uses a labeled dideoxy terminator nucleotide that has base pairing properties different from those of the modified precursor nucleotide. an unlabeled dideoxy terminator nucleotide having the same base pairing properties as the modified precursor nucleotide allows detection of the variant or mutant sites only while no non-variant sites are detected. This aspect of the invention is P1155 particularly advantageous as the detection of sequence variations alone allow the scanning and detection of very high mutation capacity and allows the identification of nucleic acids based on their sequence variations. It will be appreciated that the amplification of any White DNA, which is heterozygous for a mutation or polymorphism, generates four different duplex DNAs, that is, (taking as an example a mutation from G to A in position X in the DNA sequence), a fourth will be homoduplex of a base pair GC in position X, a fourth will be homoduplex with base pair AT in position X, a quarter will be heteroduplex with base pair GT at position X and a quarter will be heteroduplex with base pair AC at position X. Similarly, heteroduplex DNA can be easily generated by denaturing and fixing new account of two homoduplex DNAs that carry sequence differences. Thus, the nature of the 3 'end sequence of the extensible fragments can be determined by their ability to function as primers in a subsequent extension reaction, using a template nucleic acid. Essentially, this determination is based on the ability of the 3 'end of the extendable fragment to hybridize a selected template in P1155 selected conditions. After partial or total hybridization, the extendable fragment can be extended using a nucleic acid polymerase and nucleic acid precursors or combinations selected from them as described herein. It will be appreciated that there are multiple possibilities for the selection of template molecules. However, the extension of the extendable fragment is a measure of its hybridization or lack of it to a selected template molecule and, thus, the determination of the nature of the 3 'end sequence of the extendable fragments is effected. on this basis, since this 3 'sequence is indicative of the original white nucleic acid sequence. Normally, the template molecule is selected so as to carry a sequence of partial or complete complementarity to the fragment in the 5 'direction. The 5 'fragment may extend one or more nucleotides into the template molecule using a nucleic acid polymerase and nucleic acid precursors or a combination thereof or dideoxy terminator nucleotides or a combination of normal nucleic acid precursors and dideoxy terminator nucleotides . The extension of step iv) can be achieved by means of an amplification reaction using the extensible DNA fragment.

PX155 Alternatively, the extension of step iv) is achieved by means of an amplification reaction that includes a primer in addition to using the extensible DNA fragment. The repeated extension of a 5 'fragment in a template nucleic acid in combination with a second flanking primer that can be extended in the template copy allows the amplification of the template nucleic acid. These amplified products can be easily detected by standard nucleic acid staining methods, such as ethidium bromide after resolution by electrophoresis. Alternatively, the template molecule can be selected so that it can be extended using the fragment in the 5"direction as a template and so that the extension is based on hybridization at the 3 'end of the fragment in the 5' direction. 5 'direction can be analyzed based on its ability to function in a 5' nuclease assay During the extension of the fragment in the 5 'direction by a polymerase with 5' to 31 nuclease activity, the activity of the 5 'to 3' nuclease degrades an indicator molecule in the 3 'direction fixed to the same template chain as the fragment in the 5' direction.

P1155 fragment in the 5 'direction is extended in a synthetic mold containing indicator and extinction labels, then the cleavage of the resulting double-stranded DNA will release the indicator from the quencher and a signal will be detected. Normally this excision will be performed by an enzyme that recognizes the double-stranded DNA molecule. Normally, this enzyme will be a restriction enzyme. In addition, the sequence of the 3 'end of the 5' fragment can be determined on the basis of its hybridization to other nucleic acid molecules. A further possibility is that the products extended or amplified using fragments in the 5 'direction can be detected on the basis of their filtration and / or precipitation properties. When analyzing the extension and incorporation of nucleotides in step iv), where in an extension reaction fragments are used in the 5 'direction, it is important to verify that any observed extension is specifically due to the extension of the fragments in the 5' direction and it is not due to the extension of the initial primers, which were not used during the initial amplification, they were used in step i) or the extension of possible non-specific 5 'fragments that can be generated by the rupture or non-specific DNA damage during the previous steps P1155 of the procedure. To overcome this "noise", the DNA, before the glycosylase-mediated treatment, can be treated with a single-stranded specific nuclease DNA, for example, Exonuclease I, which will degrade the unused primers and the non-specific single-stranded DNA and later it can be deactivated by heat. DNA can be treated with 3 'AP endonuclease / lyase that will cut DNA and primers at any pre-existing PA sites, specifically generated or generated by DNA damage. The 3 'AP endonuclease / lyase is subsequently removed from the reaction. Because the endonuclease / lyase cleaves on the 3 'side, the resultant contaminating 5' fragments are not extensible and will not interfere with the extension of the subsequently generated 5 'fragments. In addition to these treatments, a control reaction can be performed to check non-specific extensions. Thus, in step iii) of the method, AP sites can be cut with a 3 'AP endonuclease / lyase, thereby generating fragments in the non-extensible 5' direction. However, if the extension and incorporation of nucleotides is observed in the subsequent step iv), then we can measure or determine the level of non-specific extension achieved during the procedure.

P1155 The similarity is important to ensure that the incorporation of nucleotides, labeled or untagged, in step iv), is due to the incorporation of those nucleotides supplied during step iv) and not those from a previous step. This is especially important when the reaction involves the incorporation of dideoxynucleotides. Before or after cleavage of the DNA at abasic sites and generation of fragments in the 5 'direction, the reaction can then be treated with a phosphatase that digests all unincorporated nucleotides present in the reaction, ie, alkaline phosphatase from shrimp that it can later be inactivated by heat. In a further embodiment of the invention, the enzyme used in step iv) is a ligase that can be incubated with the 5 'extendable fragment in the presence of a reporter oligonucleotide. The indicator oligonucleotide can degenerate partially. The method according to the invention can be used among others to detect a known or unknown mutation and to detect differences and similarities in the genomes. These aspects of the invention are further illustrated below. The method according to the invention provides in one aspect a means for generating primers P115S random in a simple and easy way. Essentially, the introduction of a modified base into an amplified DNA product, followed by glycosylase-mediated cleavage and subsequent treatment of the cleavage products, so that they can be extended by a nucleic acid polymerase, provides a rapid medium to generate random primers. The subsequent use of these primers, that is, the fragments extendable in the 5 'direction, in the random amplification of target nucleic acids allows the amplification of discrete DNA molecules from the target nucleic acids, thus allowing the characterization of the acid nucleic acid based on the similarities and differences of the amplified products. Since these primers are derived essentially from the target nucleic acid, specifically their 3 'ends, they are better primers for the subsequent analysis by random amplification of the target nucleic acid and the amplification is more specific. Many discrete DNA products were generated during the random amplification of nucleic acids. A discrete DNA product can be separated from other products based on size. The method according to the invention allows the generation of primers from the total or a part of the separated product. The use of an initial primer in the random amplification of P1155 a nucleic acid that allows the immobilization of the separated product, allows the isolation of the upper and lower primers extended to the first point of the cleavage mediated by glycosylase. The 3 'end of these 5' fragments are derived from the target nucleic acid and, thus, allow more specific amplification of the target nucleic acid or related nucleic acids. The 3 'end of these fragments in the 5' direction can be short or long. By "short 3" ends in the present one is referred to from one to three nucleotides, while by 3 'long ends it is referred to herein as more than three nucleotides.The longer 3' ends in these fragments in the 5 'direction are more desirable as they allow a very specific amplification of a target nucleic acid sequence The 5 'fragments generated with longer 3' ends can be selected by size classification methods Alternatively, the initial amplification primers can be designed to promote the binding of a protein that protects a section of the 3 'region of the initial extended primer.Thus, this region is refractory to glycosylase-mediated cleavage, due to protection by the protein and inclusion of this design. primer and protein allows the generation of fragments in the P1155 5 'with 3' ends longer. This embodiment of the invention provides a rapid and simple method for the generation of random and specific primers for the amplification of nucleic acid without prior knowledge of the nucleic acid sequence. It will be appreciated that the random amplification of nucleic acid using arbitrarily chosen primers is a well-known method for detecting similarities and differences between genomes. This random amplification is based on the fixation of the arbitrarily chosen primers in the target samples followed by multiple rounds of enzymatic amplification, whereby the primers are extended using the genomic DNA or cDNA selected as template. Using these primers in this method results in the amplification of discrete DNA molecules. The analysis of these molecules allows the investigation of similarities and differences between different samples. Normally, many different random primers are chosen for the investigation of a genome or cDNA and these primers were chemically synthesized and designed in a random fashion with the assumption that they will hybridize the target nucleic acid in the amplification process. The method according to the invention allows the easy and rapid generation of primers P1155 from a white nucleic acid, which can subsequently be used for the random amplification of the same or a different target nucleic acid. In addition to the extension by a polymerase reaction, as indicated above, fragments in the 5 'direction of step iv) may also be extended by the binding of another single-stranded DNA molecule resulting in extended 5' fragments. larger than the initial 5 'fragments generated by the glycosylase-mediated excision. The DNA molecule to which the fragment is ligated in the 5 'direction is referred to in the present indicator oligonucleotide and this may vary in length. The binding of the fragments 5 'to the indicator oligonucleotide depends on the attachment of both DNA molecules (the fragment in the 5' direction and the reporter oligonucleotide) to a template molecule at adjacent sites, so that the fragment ends in the direction 5 'and the indicator oligonucleotide are juxtaposed. This means that the 3 'base end of the 5' fragment is juxtaposed to the 5 'base end of the reporter oligonucleotide. The indicator oligonucleotide is usually a synthetic oligonucleotide but can also be any other type of DNA molecule or RNA molecule. The template molecule is selected so as to carry a partial or complete sequence complementarity with the fragment in the 51 direction and the reporter oligonucleotide. The template of choice may be the amplified nucleic acid originally intact or cleaved with glycosylase. In addition, the template can be a single-stranded DNA molecule, for example, a synthetic oligonucleotide, which can vary in length and which will allow the complementary attachment of the fragments in the 5 'direction and of the reporter oligonucleotides. The template DNA can be single chain or double chain in nature. The double-stranded DNA that acts as a template consists of the template chain and the complementary chain. The double-stranded DNA must first be denatured and then allowed to bind again in the presence of the 5 'fragment and the reporter oligonucleotide. The 5 'fragment and the indicator oligonucleotide then compete with the complementary strand in the binding to the template chain of the double-stranded template molecule. Several DNA ligases and RNA ligases can be used to extend the 3 'end of the extendable fragment by ligating an indicator oligonucleotide into a template nucleic acid. The DNA ligases from P1155 many sources, which include those isolated from phage, for example, T4 DNA ligase and mesophilic and thermophilic bacteria can be used to bind the indicator oligonucleotide to the extensible fragment. The thermophilic DNA ligases can be used in repeated cyclic ligations of the extensible fragments in the reporter oligonucleotides. As mentioned above, the method according to the invention includes the production of a fragment in the direction 51 having a 3 'hydroxyl group. This is an essential requirement for the extension of the molecule by the addition of nucleotides by a polymerase and also for the extension by ligating an indicator oligonucleotide by a ligase. In addition to a 3 'hydroxyl group in the fragment in the 51 direction, the indicator oligonucleotide is required to have a phosphate group 51 for binding to occur. The binding also allows the detection of the fragment in the 3 'direction if desired. The fragment in the 3 'direction is the rest of the DNA chain from which the fragment was cleaved in the 5"direction .. Here, the indicator oligonucleotide is required to have a 3' hydroxyl end and the fragment in the 3 'direction 5 'extreme phosphate group, since glycosylase-mediated cleavage can generate several different 5' fragments in one P1155 individual reaction, due to the presence of normal and mutant alleles in the target nucleic acid or due to the random introduction of modified bases, the extension by binding reaction may contain several different 5 'fragments in addition to the several different reporter oligonucleotides and template nucleic acids. In addition, several different nucleic acids can be simultaneously characterized, since an individual reporter oligonucleotide and / or a template nucleic acid can be used for the characterization of each individual nucleic acid under investigation. The fragment extended in the 5 'direction can be detected by any of several means including size analysis, hybridization and amplification. In addition, the DNA molecule resulting from the binding can be further amplified in a polymerase chain reaction. The reporter oligonucleotide, the 5 'fragment or the template nucleic acid may be labeled. For example, a fluorescent or radioactive label can be used in addition to a biotin or digoxigenin label. A useful marker in the indicator oligonucleotide is a radioactive 5 'end phosphate, ie 32P or 33P. This radioactive phosphate serves as a marker with which DNA is detected and also has the P1155 required 5 'phosphate in the reporter molecule. A marker of biotin in the reporter oligonucleotide, in the fragment in the 5 'direction or in the template nucleic acid, will serve to immobilize directly the fragment extended in the 5' direction or by hybridization, in a solid support. The immobilization, in combination with multiple different ligation extensions, will serve to produce a very efficient system with high capacity for the characterization of DNA molecules. The invention, which utilizes binding extension, can also be used to identify unknown sequence changes in the nucleic acid by its ability to identify a mutant fragment in the 5 'direction in a mixture of normal 5' fragments. A partially degenerate reporter oligonucleotide can be used in the binding reaction. Selective binding of a 5 'fragment that is caused by the cleavage of a DNA molecule at a mutation site can also be achieved by using a degenerate reporter oligonucleotide wherein the end 51 is complementary to the normal allele. In addition, the invention can be used to screen all CpG dinucleotide sites within a DNA fragment by using a completely complementary reporter oligonucleotide designed to be fixed at each CpG site. The length of P1155 any resulting binding product will indicate the CpG site that has mutated. Suitably, any extended fragments resulting from step iv are detected by hybridization. The method according to the invention offers significant advantages over existing methods in that it: a) allows the detection of similarities or differences or of similarities and differences between nucleic acid samples. In particular, it allows the detection thereof in a large number of multiple different places in the nucleic acid. While it is possible to use other methods to reach this end point, this method offers the advantage of a single process that can be easily scaled, allowing the rapid and easy characterization of nucleic acid molecules. b) It will be appreciated that nucleic acid amplification is a common method for characterizing and detecting nucleic acids. The amplification depends on the use of primers that extend in the amplification process. In addition, the method according to the invention allows the generation of high specificity primers for the amplification of nucleic acids without the need of prior knowledge of any sequence of the acid P1155 nucleic acid. In this way, the present method has a high utility in the characterization of nucleic acids by analyzing the products of the amplification generated therefrom. The amplification approaches described for characterizing nucleic acids, according to the invention, allow the characterization of nucleic acids in such a way that it was not possible before the present invention. c) In the field, there is a need for simplified methods for the detection of specific mutations in the candidate sites. The method according to the invention offers this simplified method for the detection of these mutations. In particular, the 5 'fragment generated from the sites of variation of the sequence in the nucleic acids allows the analysis of these sites using many different analytical approaches and allows the precise and simplified detection of sequence variations in these places. d) The method according to the invention offers a more reproducible method for characterizing nucleic acids by random amplification and is less susceptible to error. The method according to the invention can also be used to analyze the CpG content of DNA by detecting transitions from C to T.

P1155 With the analysis of the mutation data and the mutation spectrum that have been generated during many years in the investigation of the mutation, a defined active zone for mutations in all organisms that contain 5-methylcytosine in their genomic DNA, by For example, humans have been identified, namely mutations in CpG dinucleotides. CpG dinucleotides are a site for methylation with cytosine in human cells and have been implicated in many structural and regulatory functions in the organization of the genome and in the expression of the gene, respectively. The cytosine in DNA is usually susceptible to a low but measurable level of deamination to form uracil, an event that is mutagenic if not repaired. However, with methylation at the 5 'site, the cytosine ring is now even more susceptible to spontaneous deamination. Therefore, the methylcytosine residue is deaminated to become thymine. After deamination, the CpG dinucleotide is converted to a TpG dinucleotide. Therefore, since 5-methylcytosine occurs only in a CpG and that the main cause of the mutation in this site is due to the deamination of 5-methylcytosine, the CpG sequences with mutation appear in the DNA as sequences of TpG dinucleotide that represent a classical mutation by transition from C to T.

P1155 Since CpG has been shown to be an active zone for mutation in human genetic studies, a rapid screening for improved detection of mutations in CpG dinucleotides within the DNA test fragment is very advantageous. To improve the detection of mutations in the CpG sequences, one of the following post-amplification procedures of the target DNA can be performed, followed by cleavage to generate the fragments in the 5 'direction. Step iv) is carried out in the presence of a polymerase, dideoxy TTP (ddTTP), labeled dideoxy CTP (ddCTP) and wild-type DNA as a template, the fragments in the 5 'direction are extended by the incorporation of dideoxynucleotides but only in the sites where C has been mutated to T, including the sites where CpG has mutated to TpG will be marked after the incorporation of the labeled ddCTP. Since ddCTP is a chain terminator nucleotide, DNA will not be extended beyond this point. Therefore, the DNA will be extended in mutated CpG sequences and will also be labeled and detectable. Step iv) is carried out in the presence of a polymerase, wild-type DNA as template, dTTP and dideoxy GTP (ddGTP) labeled, the fragments in the 5 'direction are extended by the incorporation of dTMP, but, P1155 only the sites where T is followed by G, that is, the TpG sequences will be marked after the incorporation of the labeled ddGTP. Since the ddGTP is a chain terminator nucleotide, the DNA will not extend beyond this point. Therefore, DNA is now extended in all TpG sequences and is also labeled and detectable. Since TpG dinucleotides occur naturally in DNA as they arise from CpG deamination, wild type DNA will show a characteristic band pattern (normally only a few bands will be observed). The label can be, for example, fluorescent 33P radioactive or biotin labeled in a manner known per se. Step iv) is carried out in the presence of polymerase, dUTP, dGTP, dATP, labeled dCTP and wild-type DNA as a template, fragments in the direction 51 extend by the incorporation of deoxynucleotides and remain labeled. The fragments 'extended in the 5' direction are subsequently cleaved by uracil-DNA glycosylase and an abasic site cleavage agent. Only the 5 'fragment that was generated by cleavage at the mutation site will remain extended and marked. This procedure will detect all mutations that generate a T incorporation site, including mutations from C to T in the CPG sites. This procedure P1155 has the advantage that step iv) utilizes the incorporation of deoxynucleotides that are incorporated more efficiently than the dideoxynucleotides by all polymerases. In addition, in heterozygous samples, the CpG with mutation will give rise to T / G (or U / G after the introduction of dUTP as a modified nucleotide). Thymine-DNA glycosylase can be used to cleave specifically in these T / G or U / G discrepancies in a CpG sequence with mutation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of an AP site generated during a step of the method according to the invention and of several ways in which the DNA at the abasic site can be cleaved to generate a fragment of DNA in the 5 'direction that can be extended; Figure 2 is a schematic representation of the method according to the invention, as described in Example 1; Figure 3 is a schematic representation of the method according to the invention, as described in Example 2, where a labeled ddTTP was used in a linear amplification reaction after the generation of an extendable fragment in the 5 'direction, - P1155 Figure 4 is a schematic representation of the method according to the invention, as described in Example 2, where a labeled ddCTP was used in a linear amplification reaction after the generation of a 5 'extendable fragment; Figure 5 is a schematic representation of the method according to the invention as described in Example 3; Figure 6A-6D is a schematic representation of the extension products obtained in Example 3, after electrophoresis and autoradiography and analysis of autoradiographs; and Figures 7A-7D are a schematic representation of the binding reactions performed in Example 4 on the fragments in direction 51 and the products obtained in this way. Figure 1 represents a single DNA strand with 5 'to 3' orientation. The position of two bases is shown, that is, base 1 and base 3. Base 2 has been eliminated and in this position there is an AP site. The vertical lines denote the ribose ring that is attached to the base. The diagonal lines with 'P1 in a circle refer to the phosphodiester bonds that bind each ribose. The DNA can be cut on the 5 'side or on the 3' side of an AP site, as shown by the arrows.

P1155 If the DNA was cleaved on the 3 'side of the AP site, ie the Z position, then the 5' fragment (containing the base 1) has a deoxyribose phosphate portion at its 3 'end as shown in box C. Cleavage of DNA on the 31st side of the AP site can be achieved by treatment with a 3 'AP endonuclease / lyase or heat treatment as described above. The AP site can also be cut in two different ways on the 5 'side of the AP site, ie, at the X and Y positions. The cleavage at the X position results in an OH group at the 3' end on the fragment in the direction 5 ', as shown in box A. Cleavage at position X can be achieved by treatment with 5' AP endonuclease, as described above. The cleavage at the Y position results in a phosphate group at the 3 'end of the fragment in the 5' direction, as shown in box B. The cleavage at the Y position can be achieved by heat and alkali treatment as described above. The invention will be further illustrated by the accompanying Examples.

P11S5 FORMS FOR CARRYING OUT THE INVENTION Example 1 The method according to the invention was used to demonstrate the production of an extended DNA fragment by extending from the 3 'OH group a fragment in the 5' direction, which has been generated by cleaving DNA containing uracil at the site of incorporation of the modified nucleotide. The target nucleic acid was a region of the RYR1 gene (952 to 1044) amplified from the human cDNA using the upper (952 to 972) and lower (1024 to 1044) primers to generate double column DNA of 93bp in length. (The nucleotide numbers refer to the sequence of the RYR1 gene). Figure 2 is a schematic diagram of the target nucleic acid and the upper and lower primers (the primers contain standard bases, G, A, T and C). Six pmoles of the lower primer was labeled at the end by incubation with 1 unit of T4 polynucleotide kinase (which is available commercially from New England Biolabs), 70mM Tris-HCl (pH7.6), MgCl2 10M, 5mM dithiothreitol and 32P ATP IμCi (3000Ci / mmol) for 30 minutes at 37 ° C. The target nucleic acid sample was amplified by PCR in a reaction mixture containing white nucleic acid, dATP, dCTP, dGTP and 0.2mM dUTP, 6pmoles of the lower primer labeled with 32P and upper primer not labeled in a P1155 total volume of 19μl. The reaction mixture was then covered with an equal volume of mineral oil and a warm start PCR was performed whereby the reaction mixture was heated at 94 ° C for 5 minutes before the addition of 1 unit of Taq polymerase ( that can be obtained from Promega) (bringing the total volume to 20μl). Thirty denaturing, fixation and extension cycles were performed in a thermocycler. The reaction mixture carrying the amplified white nucleic acid was then treated with exonuclease I (which can be obtained from Amersham Life Sciences) to digest non-extended primers in the step of amplification and shrimp alkaline phosphatase (SAP for its acronym in English) (which can be obtained from Boehringer Mannheim) to digest unincorporated dNTPs during the amplification step. This was achieved by incubating lOμl of the PCR reaction with 0.5 units of exonuclease I and 1 unit of SAP at 37 ° C for 30 minutes. Exo I and SAP were subsequently inactivated with heat by incubating the reaction at 80 ° C for 15 minutes. The uracil-DNA glycosylase (available from New England Biolabs). (0.5 units) was then added and the incubation continued at 37 ° C for 30 min. After treatment with uracil-DNA glycosylase, the abasic sites generated in the amplified product are P1155 cleaved to completion by adding NaOH to a final concentration of 0.05M and heating the mixture for 15 min at 95 ° C. The digested DNA was then precipitated by adding 10% by volume of 3M sodium acetate and 2 volumes of ethanol. The granule was resuspended in 5μl of water. The digested DNA was then treated with 0.5 units of T4 polynucleotide kinase (PNK) which removes the phosphate group from the 3 'ends. A linear amplification reaction was then carried out using the products of the above cleavage reaction, the fragment of interest which will be the labeled 5 'extendable fragment, during which the extendable fragment in the 5' direction is extended by a DNA polymerase thermostable in a cyclization reaction in a total volume of lOμl. The template for this reaction is amplified white nucleic acid (952 to 1044 of the RYR1 gene) that is free of primers due to pretreatment with Exol. To the sample an equal volume of formamide loading dye (90% formamide, 0.025% bromophenol blue, 0.025% xylene cilanol) was added and then heated at 85 ° C for 5 minutes. The sample was then loaded on a 20% denaturing polyacrylamide gel (7M urea) and the electrophoresis was carried out for 3 to 4 hours at 60W for the analysis of the size of the products P -.-. 55 extension. After electrophoresis, autoradiography was performed by exposing the gel directly to a photographic X-ray film for 12 hours at 70 ° C. Analysis of the autoradiography, where the lower primer was marked, showed a product of 93 nucleotides in length. This product was not observed if the treatment with T4 polynucleotide kinase or the linear amplification reaction was not included in the previous procedure.

Example 2 The method according to the invention was used to detect the presence of a mutation from G to A at position 1021 in the human RYRl gene. The cDNA of a normal individual and that of an individual affected by Malignant hyperthermia was amplified using primers (952 to 972) and lower (1204 to 1224) to generate a double-stranded DNA fragment of 273 base pairs (952 to 1224) (as in the case of Example 1, the nucleotide numbers refer to the sequence of the RYR1 gene). Figures 3 and 4 are schematic diagrams of the target nucleic acid and the top and bottom primers (the primers contain standard bases G, A, T and C). The target nucleic acid sample was amplified by PCR in an acid-containing reaction mixture P1155 white nucleus, dATP, dCTP, 0.2mM dGTP and 0.19mM dTTP and O.OlmM dUTP and 6pmoles of upper and lower primers in a total volume of 19μl. The reaction mixture was then covered with an equal volume of mineral oil and a warm start PCR was carried out, whereby the reaction mixture was heated at 9 ° C for 5 minutes, before the addition of 1 unit of Taq. polymerase (bringing the total volume to 20μl). Thirty denaturing, fixation and extension cycles were performed in a thermocycler. The reaction mixture carrying the amplified white nucleic acid was then treated with exonuclease I to digest the primers not extended in the amplification step and the shrimp alkaline phosphatase (SAP) to digest the unincorporated dNTPs during the passage of the amplification. This was achieved by incubating lOμl of the PCR reaction with 0.5 units of exonuclease I and 1 unit of SAP at 37 ° C for 30 minutes. Exo I and SAP were thermally inactivated subsequently by incubating the reaction at 80 ° C for 15 minutes. The uracil-DNA glycosylase (0.5 units) was then added and the incubation continued at 37 ° C for 30 minutes. After treatment with uracil-DNA glycosylase, the abasic sites generated in the amplified product were cleaved to completion by the P1155 addition of NaOH to a final concentration of 0.05M and heating the mixture for 15 minutes at 95 ° C. The digested DNA was then precipitated by adding 10% by volume of 3M sodium acetate and two volumes of ethanol. The granule was resuspended in 5μl of water. The digested DNA was then treated with 0.5 units of T4 polynucleotide kinase, which removed the phosphate group from the 3 'ends. A linear amplification reaction was then performed using the products of the above cleavage reaction, i.e., the various extendable fragments in the 5 'direction during which the fragment in the 51 direction is extended by a thermostable DNA polymerase (ie, Thermosequenase). (obtainable from Amersham Life Sciences)) in a cyclization reaction in a total volume of 10 μl. The template for this reaction is the amplified fragment of the normal cDNA (952 to 1224 of the RYR1 gene) that is free of unlabeled primers due to pretreatment with Exol. The extension reaction was carried out in the presence of lmM of three of the dideoxy terminator nucleotides (ddNTP) and 0.02mM of a ddNTP labeled with 33P. An equal volume of formamide loading dye (90% formamide, 0.025% bromophenol blue, 0.025% xylene cilanol) was added to the sample, which was then heated at 85 ° C for 5 minutes. The sample P-.155 was then loaded on a 6% denaturing polyacrylamide gel (7M urea) and electrophoresed for 3 to 4 hours at 60W for the size analysis of the extension products. After electrophoresis, autoradiography was performed by exposing the gel directly to a photographic X-ray film for 12 hours at -70 ° C. Analysis of the autoradiography, where the ddTTP was marked with ddNTP (Figure 3), showed a ladder of marked fragments corresponding to the distance of the 5 'end of the primers to the site of a dUMP incorporation (only shown the extension of the lower chain). These sites corresponded to the wild-type T-pattern of the DNA. When mutant white nucleic acid was used to generate the extension primers, an additional band was observed in the T band pattern (203 nucleotides). Analysis of the autoradiography, where the ddCTP was marked (Figure 4) showed no marked bands when the amplified normal white nucleic acid was the source of the fragments in the 5 'direction, however, when mutant white nucleic acid was used amplified, the analysis showed only one band. The size of the labeled fragment corresponds to the distance between the 5 'end of the lower primer and the mutation site (ie, 203 nucleotides) demonstrating P1155 in this way the presence of a mutation from G to A at position 1021 of the RYRl gene in that individual.

Example 3 The method according to the invention was used to detect the presence of a mutation from G to A at position 6411 (codon 12) in the human Ki-ras gene. Genomic DNA from normal tissue and tumor tissue of an individual with colon cancer was amplified using the upper (6390 to 6409) and lower (6417 to 6443) primers to generate a 54-pair double-stranded DNA fragment. of bases (from 6390 to 6443) (the nucleotide numbers refer to the genomic sequence of the Ki-ras gene including introns). Figure 5 is a schematic diagram of the target nucleic acid and the upper and lower primers (the primers contain the standard bases G, A, T and C). The target nucleic acid sample was amplified by PCR in a reaction mixture containing white nucleic acid, dATP, dCTP, dGTP and 0.2mM dUTP and dpmoles of upper and lower primers in a total volume of 19μl. The reaction mixture was then covered with an equal volume of mineral oil and a hot start PCR was carried out whereby the reaction mixture was heated at 94 ° C for 5 minutes before the addition of a Taq unit.

P1-.55 polymerase (bringing the total volume to 20μl). Thirty denaturing, fixation and extension cycles were performed in a thermocycler. The reaction mixture carrying the amplified white nucleic acid was then treated with Exonuclease I (Exo I) to digest the primers that did not spread in the amplification step and shrimp alkaline phosphatase (SAP) to digest unincorporated dNTPs during the step of amplification. This was achieved by incubating 20μl of the PCR reaction with 0.5 units of Exo I and 1 unit of SAP at 37 ° C for 30 minutes. Exo I and SAP were thermally inactivated subsequently by incubating the reaction at 80 ° C for 15 minutes. Uracil-DNA glycosylase (0.5 units) and Endonuclease IV (1 unit) were then added and incubation continued at 37 ° C for 30 minutes to allow total cleavage of all uracils present in the amplified DNA and excision of the resulting abasic sites until their completion. This cleavage results in 5 'fragments having a 3' hydroxyl group at their 3 'ends that can be extended by the action of a DNA polymerase. The extension reactions were then carried out in a reaction mixture (10 μl) containing the products of the above cleavage reaction, ie the extensions extendable in the 5 'direction (2 μl reaction).

Cleavage P1155, approximately lpmol of extensible fragments) and lOOfmol of a synthetic template oligonucleotide, dATP, dTTP and 0.2mM dGTP, 0.02mM dCTP, dCTP lCia32P, 6pmol of the imterso primer and 1 unit of Taq polymerase DNA. The reaction was carried out for 40 cycles denaturant, fixation and extension. To the sample an equal volume of formamide loading dye (90% formamide, 0.025% bromophenol blue, 0.025% xylene cilanol) was added, which was then heated at 85 ° C for 5 minutes. The sample was then loaded on a denaturing polyacrylamide gel (7M urea) and the electrophoresis was carried out in 3 to 4 hours at 60W for size analysis of the extension products. After electrophoresis, autoradiography was performed by exposing the gel directly to a photographic X-ray film for 12 hours at -20 ° C. Analysis of normal tissue DNA results in the generation of an extensible fragment of 37 nucleotides after glycosylase-mediated cleavage using the above-mentioned higher and lower primers (Figure 5). A similar analysis of the DNA of tissue with tumor results in the generation of an extensible fragment of 32 nucleotides (Figure 5). Analysis of the autoradiography showed a band of 66 nucleotides after DNA analysis of normal tissue and using oligonucleotide 1 template (Figure 6A). This band was not observed when oligonucleotide 2 template was used in the previous analysis (Figure 6B). Analysis of the autoradiography showed a band of 66 nucleotides after DNA analysis of the tumor tissue and template oligonucleotide 2 was used (Figure 6C). This band was not observed when Oligonucleotide 1 template was used in the previous analysis (Figure 6D). Thus, the presence of a mutation in codon 12 of the Ki-ras gene was determined by the ability of the fragment in the 5 'direction to spread in a mutant template oligonucleotide, whereas the fragment in the 5' direction did not extend in a template oligonucleotide normal and vice versa for the absence of the mutation.

Example 4 Example 3 was repeated until the stage where the abasic sites were cleaved to completion. This cleavage resulted in 5 'fragments having a 3' hydroxyl group at their 3 'ends as before. Reporter oligonucleotides 1 and 2 (Figure 7) (complementary to nucleotides 6397 to 6406 (5'TCCAACTACC3 'No. 1 (SEQ ID NO: 28)) and nucleotides 6402 to 6411 (5' CCAGCTCCAA3 ', No. 2 ( SEQ ID NO: 30)) of the Ki-ras gene, respectively), where the 5 'end labeled P1155 using ATP? 32P and T4 polynucleotide kinase. This served to mark the resulting ligated fragment and also provided a 5 'end phosphate in the indicator oligonucleotide that is required for any possible binding. The binding reactions (16 ° C for 60 minutes) were then carried out using the products of the above cleavage reaction, ie, the 5 'extendable fragments (5μl of the cleavage reaction, approximately 2pmol of extendable fragments), 4 pmol of the labeled indicator oligonucleotide and 2 pmol of an oligonucleotide 1 or 2 template (5 * GGTAGTTGGAGCTGGTGGCG3 '(SEQ ID No. 27) (nuc 6397 to 6416) or 5' TTGGAGCTGGTGGCGTAGGC3 '(SEQ ID No. 31) (nuc 6402 to 6421) , respectively) (Figure 7) and 1 unit of T4 DNA ligase, during which the fragment in the 5 'direction was extended in its length by the binding of an indicator oligonucleotide in 20 μl of reaction. To the sample an equal volume of formamide loading dye (90% formamide, 0.025% bromophenol blue, 0.025% xylene cilanol) was added, which was then heated at 85 ° C for 5 minutes. The sample was then loaded on a denaturing polyacrylamide gel (7M urea) and the electrophoresis was performed from 3 to 4 hours at 60W for size analysis of the extension products. After electrophoresis, autoradiography was performed by exposing the gel directly to a photographic X-ray film for 3 hours at -20 ° C. Analysis of normal tissue DNA results in the generation of an extensible fragment of 37 nucleotides after glycosylase-mediated cleavage, using the aforementioned upper and lower primers (Figure 5). A similar analysis of the DNA of tissue with tumor results in the generation of an extensible fragment of 32 nucleotides (Figure 5). Analysis of the autoradiography showed a band of 47 nucleotides after DNA analysis of normal tissue and using oligonucleotide 1 indicator and oligonucleotide 1 template (Figure 7A). This band was not observed when oligonucleotide 2 indicator was used in the previous analysis (Figure 7B). Analysis of the autoradiography showed a band of 42 nucleotides after DNA analysis of the tissue with tumor and using oligonucleotide 2 indicator and oligonucleotide 2 template (Figure 7C). This band was not observed when oligonucleotide indicator 1 was used in the previous analysis (Figure 7D). Therefore, the presence of a mutation in codon 12 of the Ki-ras gene was determined by the presence of a band of 42 nucleotides, while the presence of the normal allele was determined by the presence of a band of 47 nucleotides. Samples containing normal DNA and P1155 tumor produced both bands of 42 and 47 nucleotides. The above analysis was also performed where the lower primer was labeled at the 5 'end with 32P. This required that reporter oligonucleotides be phosphorylated using unlabeled ATP as a phosphate donor. The results were similar to those previously observed in that a 42n band demonstrated the presence of a mutant Ki-ras gene (codon 12), while a 47n band demonstrated the absence of the mutation in codon 12 of the Ki-ras gene . In addition, the above analysis was also performed using the Ki-ras gene fragment initially amplified from the normal or mutant sample as a template during the binding reaction. The normal amplified product was used instead of the oligonucleotide 1 template, whereas a mutant amplified product was used instead of the template oligonucleotide 2. Again, the same results as those described above were observed. As indicated above, the method according to the invention has numerous advantages over known methods, especially the method of WO 97/03210. After glycosylase-mediated cleavage in the case of WO 97/03210 (which can be performed in various forms and which produces several different 3 'terminals), the resulting DNA fragments are not P1155 further processed and analyzed directly. In the present invention, the step of glycosylase-mediated cleavage followed a step allowing expansion of the 3 'terminals generated by glycosylase-mediated cleavage. A main advantage of the present invention, which is not possible in the case of the methods of the prior art, is that the present invention allows the detection of sequence differences, such as mutations and polymorphisms between nucleic acid molecules without detecting similarities of sequence. As indicated above, it is not possible with the method of WO 97/03210 to detect sequence differences between nucleic acid molecules without detecting sequence similarities. This is a limitation of the method of WO 97/03210, since multiple samples can not be combined for simultaneous analysis. The present invention also allows the analysis of multiple genes or the simultaneous analysis of gene segments. Furthermore, the present invention allows the generation of specific primers for the amplification of nucleic acids without the need to have prior knowledge of the nucleic acid sequence. This is not possible with the known methods. In addition, the present invention allows the generation of specific primers of nucleic acids in a form P1155 and these primers can subsequently be evaluated by polymerase extension to determine the nature of the sequence at the 3 'terminals of the primers.

P1155 SEQUENCE LISTS (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: FORBAIRT (doing business as BioResearch Ireland) (B) STREET: Glasnevin (C) CITY: Dublin 9 (E) COUNTRY: Ireland (F) ) POSTAL CODE: No code (A NAME: UNIVERSITY COLLEGE CORK (B STREET: College Road (C CITY: Cork (E COUNTRY: Ireland (F) ZIP CODE: No code (A NAME: McCARTHY, Thomas Valentine (B STREET: Vista Villa, Montenotte (C CITY: Cork (E COUNTRY: Ireland (F) ZIP CODE: No code (A NAME: VAUGHAN, Patrick Martin (B STREET: 175 West Avenue, Parkgate, Frankfield (C CITY: Cork (E COUNTRY: Ireland (F) ZIP CODE: No code (ii) TITLE OF THE INVENTION: A method for the characterization of nucleic acid molecules, which includes the generation of DNA fragments that can extend in the 5 'direction, resulting from nucleic acid cleavage at an abasic site.

EffiÜ ^ (iii) NUMBER OF SEQUENCES: 32 (iv) FORM FOR COMPUTER READING: (A) TYPE OF MEANS: flexible co (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (F) TYPE OF TISSUE: Skeletal muscle (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1 : TCCAAGGAGA AGCTGGATGT GGCCCCCAAG CGGGATGTGG AGGGCATGGGTGAG 60 ATCAAGTACG GGGAGTCACT GTGCTTCGTG CAG 93 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear P1155 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by PCR amplification" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: TCCAAGGAGA AGCTGGATGT GGCCCCCAAG CGGGAUGUGG AGGGCAUGGG CCCCCCUGAG 60 AUCAAGUACG GGGAGUCACU GUGCUUCGUG CAG 93 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: l ineal (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by PCR amplification" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: CTGCACGAAG CACAGTGACT CCCCGUACUU GAUCUCAGGG GGGCCCAUGC CCUCCACAUC 60 CCGCUUGGGG GCCACAUCCA GCUUCUCCUU GGA 93 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple P1155 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage and has a 3 'phosphate group" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: CTGCACGAAG CACAGTGACT CCCCG 25 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage and has a 3 'hydroxyl group" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: CTGCACGAAG CACAGTGACT CCCCG 25 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 base pairs (B) TYPE: nucleic acid P1155 (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage followed by extension of the fragment in direction 5 '"(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: CTGCACGAAG CACAGTGACT CCCCGTACTT GATCTCAGGG GGGCCCATGC CCTCCACATC 60 CCGCTTGGGG GCCACATCCA GCTTCTCCTT GGA 93 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 273 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (F) TISSUE TYPE: skeletal muscle (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7 : TCCAAGGAGA AGCTGGATGT GGCCCCCAAG CGGGATGTGG AGGGCATGGGTGAG 60 ATCAAGTACG GGGAGTCACT GTGCTTCGTG CAGCATGTGG CCTCAGGACT GTGGCTCACC 120 TATGCCGCTC CAGACCCCAA GGCCCTGCGG CTCGGCGTGC TCAAGAAGAA GGCCATGCTG 180 CACCAGGAGG GCCACATGGA CGACGCACTG TCGCTGACCC GCTGCCAGCA GGAGGAGTCC 240 CAGGCCGCCC GCATGATCCA CAGCACCAAT GGC 273 P1155 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 273 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANT I- SENSE: NO (vi) ORIGINAL SOURCE: (F) TYPE OF TISSUE: skeletal muscle (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO : 8: TCCAAGGAGA AGCTGGATGT GGCCCCCAAG CGGGATGTGG AGGGCATGGGTGAG 60 ATCAAGTACA GGGAGTCACT GTGCTTCGTG CAGCATGTGG CCTCAGGACT GTGGCTCACC 120 TATGCCGCTC CAGACCCCAA GGCCCTGCGG CTCGGCGTGC TCAAGAAGAA GGCCATGCTG 180 CACCAGGAGG GCCACATGGA CGACGCACTG TCGCTGACCC GCTGCCAGCA GGAGGAGTCC 240 CAGGCCGCCC GCATGATCCA CAGCACCAAT GGC 273 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 196 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage and fragment extension in the 5 'direction and has a 3' hydrogen atom" P1155 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: modified_base (B) LOCATION: 196 (D) OTHER INFORMATION: / mod_base = OTHER / note = "Dideoxy T "(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GCCATTGGTG CTGTGGATCA TGCGGGCGGC CTGGGACTCC TCCTGCTGGC AGCGGGTCTC 60 CGACAGTGCG TCGTCCATGT GGCCCTCCTG GTGCAGCATG GCCTTCTTCT TGAGCACGCC 120 GAGCCGCAGG GCCTTGGGGT CTGGAGCGGC ATAGGTGAGC CACAGTCCTG AGGCCACATG 180 CTGCACGAAG CACAGT 196 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 200 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: l ineal (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage followed by extension of the fragment in the 5 'direction and has a 3' hydrogen atom" (iii) HYPOTHETICAL : NO (ix) FEATURES: (A) NAME / KEY: modified_base (B) LOCATION: 200 (D) OTHER INFORMATION: / mod_base = OTHER / note = "Didesoxi T" P1155 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: GCCATTGGTG CTGTGGATCA TGCGGGCGGC CTGGGACTCC TCCTGCTGGC AGCGGGTCTC 60 CGACAGTGCG TCGTCCATGT GGCCCTCCTG GTGCAGCATG GCCTTCTTCT TGAGCACGCC 120 GAGCCGCAGG GCCTTGGGGT CTGGAGCGGC ATAGGTGAGC CACAGTCCTG AGGCCACATG 180 CTGCACGAAG CACAGTGACT 200 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 204 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage followed by extension of the fragment in the 5 'direction and has a hydrogen atom 3'" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURES: (A) NAME / KEY: modified_base (B) LOCATION: 204 (D) OTHER INFORMATION: / note = "Dideoxy T" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: GCCATTGGTG CTGTGGATCA TGCGGGCGGC CTGGGACTCC TCCTGCTGGC AGCGGGTCTC 60 CGACAGTGCG TCGTCCATGT GGCCCTCCTG GTGCAGCATG GCCTTCTTCT TGAGCACGCC 120 GAGCCGCAGG GCCTTGGGGT CTGGAGCGGC ATAGGTGAGC CACAGTCCTG AGGCCACATG 180 CTGCACGAAG CACAGTGACT CCCT 204 P1155 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 206 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage followed by extension of the fragment in the 5 'direction and has a 3' hydrogen atom" (iii) HYPOTHETICAL : NO (iv) ANTI-SENSE: NO (ix) FEATURES: (A) NAME / KEY: modified_base (B) LOCATION: 206 (D) OTHER INFORMATION: / note = "Dideoxy T" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: GCCATTGGTG CTGTGGATCA TGCGGGCGGC CTGGGACTCC TCCTGCTGGC AGCGGGTCTC 60 CGACAGTGCG TCGTCCATGT GGCCCTCCTG GTGCAGCATG GCCTTCTTCT TGAGCACGCC 120 GAGCCGCAGG GCCTTGGGGT CTGGAGCGGC ATAGGTGAGC CACAGTCCTG AGGCCACATG 180 CTGCACGAAG CACAGTGACT CCCCGT 206 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 209 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid P1155 (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage followed by extension of the fragment in the 5 'direction and has a hydrogen atom 3'" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURES: (A) NAME / KEY: modified_base (B) LOCATION: 209 (D) OTHER INFORMATION: / mod_base = OTHER / note = "DIDSOXY T" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: GCCATTGGTG CTGTGGATCA TGCGGGCGGC CTGGGACTCC TCCTGCTGGC AGCGGGTCTC 60 CGACAGTGCG TCGTCCATGT GGCCCTCCTG GTGCAGCATG GCCTTCTTCT TGAGCACGCC 120 GAGCCGCAGG GCCTTGGGGT CTGGAGCGGC ATAGGTGAGC CACAGTCCTG AGGCCACATG 180 CTGCACGAAG CACAGTGACT CCCCGTACT 209 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 204 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA generated by glycosylase-mediated cleavage followed by extension of the fragment in the 5 'direction and has a hydrogen atom 3'" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO P1155 (ix) FEATURES: (A) NAME / KEY: modified_base (B) LOCATION: 204 (D) OTHER INFORMATION: / mod_base = OTHER / note = "DIDSOXY C" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14 : GCCATTGGTG CTGTGGATCA TGCGGGCGGC CTGGGACTCC TCCTGCTGGC AGCGGGTCTC 60 CGACAGTGCG TCGTCCATGT GGCCCTCCTG GTGCAGCATG GCCTTCTTCT TGAGCACGCC 120 GAGCCGCAGG GCCTTGGGGT CTGGAGCGGC ATAGGTGAGC CACAGTCCTG AGGCCACATG 180 CTGCACGAAG CACAGTGACT CCCC 204 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: AACTTGTGGT AGTTGGAGCT GGTGGCGTAG GCAAGAGTGC CTTGACGATA CAGC 54 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid P1155 (A) DESCRIPTION: / desc = "Generated by PCR amplification of genomic DNA" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: AACTTGTGGT AGTTGGAGCT GGUGGCGUAG GCAAGAGUGC CUUGACGAUA CAGC 54 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by PCR amplification of genomic DNA" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: GCTGTATCGT CAAGGCACTC TTGCCTACGC CACCAGCUCC AACUACCACA AGUU 54 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by PCR amplification of genomic DNA" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: AACTTGTGGT AGTTGGAGCT GAUGGCGUAG GCAAGAGUGC CUUGACGAUA CAGC 54 P1155 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by PCR amplification" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 19: GCTGTATCGT CAAGGCACTC TTGCCTACGC CAUCAGCUCC AACUACCACA AGUU 54 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by glycosylase-mediated cleavage of DNA amplified by PCR" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: GCTGTATCGT CAAGGCACTC TTGCCTACGC CACCAGC 37 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear P1155 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by glycosylase-mediated cleavage of DNA amplified by PCR" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 21: GCTGTATCGT CAAGGCACTC TTGCCTACGC CA 32 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Synthetic oligonucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 22: GCTGTAAACG ACGGCCAGTT TCATGCAGGG CTGGAGTCGT AGGCAAGAGT GCCTTGACGA 60 TACAGC 66 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" P1155 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: GCTGTAAACG ACGGCCAGTT TCAT 24 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by extension with primer" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: GCTGTATCGT CAAGGCACTC TTGCCTACGC CACCAGCCCT GCATGAAACT GGCCGTCGTT 60 TACAGC 66 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 25: GCTGTAAACG ACGGCCAGTT TCATGCAGGA TCCATGGCGT AGGCAAGAGT GCCTTGACGA 60 TACAGC 66 P1155 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by extension with primer" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 26: GCTGTATCGT CAAGGCACTC TTGCCTACGC CATGGATCCT GCATGAAACT GGCCGTCGTT 60 TACAGC 66 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: GGTAGTTGGA GCTGGTGGCG 20 (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple P1155 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 28: TCCAACTACC 10 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by the binding of two DNA molecules" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: GCTGTATCGT CAAGGCACTC TTGCCTACGC CACCAGCTCC AACTACC 47 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 30: CCAGCTCCAA 10 P1155 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 31: TTGGAGCTGG TGGCGTAGGC 20 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Generated by the binding of two DNA molecules" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32: GCTGTATCGT CAAGGCACTC TTGCCTACGC CACCAGCTCC AA 42 P1155

Claims (24)

1. CLAIMS; A method for characterizing nucleic acid molecules, comprising the steps of: i) introducing into a DNA molecule a modified base which is the substrate of a DNA glycosylase; ii) cleave the modified base by means of DNA glycosylase, so that an abasic site is generated; iii) cleaving the DNA at the abasic site so that a DNA fragment is generated in the 5 'direction, which can be extended; and iv) incubating the extensible fragment in the 5 'direction in the presence of an enzyme allowing the extension thereof and of a template nucleic acid and analyzing the resulting fragments.
2. 2. A method according to claim 1, wherein the fragment in the 5 'direction is generated by cleaving the DNA on the side 51 of the abasic site, such that the 3' end of the fragment in the 5 'direction carries a group hydroxyl
3. 3. A method according to claim 2, wherein the cleavage is achieved with a 5 'AP endonuclease.
4. 4. A method according to claim 1, wherein the fragment in the 5 'direction is generated by cleavage at the 5' side of the abasic site, so that at the terminal end 31 of the fragment in the 5 'direction leave a P11S5 phosphate group and the elimination of the phosphate group, so that the fragment in the 5 'direction carries a hydroxyl group at the 3' end.
5. 5. A method according to claim 1, wherein the fragment in the 5 'direction is generated by cleavage on the 3' side of the abasic site, so as to generate a phosphate dexosirribose group at the 3 'end of the fragment in the 5-direction. and, subsequently, removing the deoxyribose group to leave a hydroxyl group at the 3 'terminal end.
6. 6. A method according to any one of the preceding claims, wherein the 5 'deoxyribose portions of the 3' end of the fragment in the 5 'direction are removed, so that the fragment in the 5' direction can be extended in the template.
7. 7. A method according to claim 6, wherein the 5 'deoxyribose portions are removed by a 5' deoxyribophosphodiesterase.
8. 8. A method according to any of the preceding claims, wherein the modified base is introduced by the enzymatic amplification of the DNA.
9. 9. A method according to claim 8, wherein the amplified chains are separated for a separate analysis of the respective chains.
10. 10. A method according to claim 8 or 9, in P1155 where the primer or the one or more nucleotides involved in the enzymatic amplification are labeled.
11. 11. A method according to any of the preceding claims, wherein the enzyme is a polymerase.
12. 12. A method according to claim 11, wherein the fragment extendable in the 5 'direction is incubated with the polymerase in the presence of 1 or more nucleotides.
13. 13. A method according to claim 12, wherein one or more of the nucleotides of step iv) is a dideoxy nucleotide.
14. 14. A method according to claims 12 or 13, wherein the one or more nucleotides of step iv) are labeled.
15. 15. A method according to any of claims 11 to 14, wherein the extension of step iv) is achieved by means of an amplification reaction using the stretchable DNA fragment.
16. 16. A method according to any of claims 11 to 15, wherein the extension of step iv) is achieved by means of an amplification reaction that includes a primer in addition to using said extensible DNA fragment.
17. 17. A method according to any of claims 1 to 10, wherein the enzyme is a ligase. P1155
18. 18. A method according to claim 17, wherein the 5 'extendable fragment is incubated with the ligase in the presence of a reporter oligonucleotide.
19. 19. A method according to claim 18, wherein the reporter oligonucleotide is partially degenerate.
20. A method according to any one of the preceding claims, wherein any of the extended fragments resulting from step iv) are detected by hybridization.
21. 21. A method according to any of the preceding claims, which is used to detect a known or unknown mutation.
22. 22. A method according to claim 1 for the characterization of a nucleic acid molecule, practically as described and exemplified in the foregoing.
23. 23. A method according to any of claims 1 to 20, wherein the method is used to analyze the CPG content of the DNA by detecting transitions from C to T in the DNA.
24. 24. A method according to claim 1, for the characterization of nucleic acid molecule, practically as described and exemplified in the foregoing. P1155