Patent Application
20100120036
The present invention relates to a method for amplifying a DNA fragment and a method for measuring the degree of methylation of a genomic DNA.
Methylation which occurs at the 5th position of cytosine (C) in a CpG dinucleotide is the only modification observed in a genomic DNA of vertebrates including mammals and is strongly involved in regulation of gene activities. Specifically, chromatins are condensed by DNA methylation, resulting in inhibition of gene activities.
Genome-wide analyses of DNA methylation status in tissues and cells, which vary depending on the tissue-types and the development stages, with a focus on the CpG islands have revealed that a tissue-dependent differentially methylated region (T-DMR), a region showing a methylation status dependent to a tissue or a cell, exists in a genic region expressed in a tissue or a cell-type specific manner, and the methylation status of T-DMR is closely associated with regulation of tissue- or cell-specific gene expression. It is important for normal development and cell differentiation to establish and maintain a normal cell-type or tissue specific DNA methylation profile consisting of many patterns of the T-DMR methylation status in the whole genome (Shiota 2004, Cytogenet Genome Res, 105, 325-34).
Analyses of the methylation status in cancer suppressor genes using cancer cells or patient specimens have indicated that abnormal methylation should be one of major carcinogenesis mechanisms. Furthermore, it has been reported that abnormality in DNA methylation is not observed in genes associated with specific carcinogenesis alone, but, in cancer cells and cell groups in the stage before carcinogenesis, the overall methylation state of a genome DNA is in an abnormal modification state as hypomethylation. Furthermore, abnormal DNA methylation has been confirmed at an inflammation site in infection shown to be associated with carcinogenesis (Maekita, Nakazawa et al. 2006, Clin Cancer Res, 12, 989-95).
As described above, the DNA methylation profile of the whole genome differs between a normal cell and an abnormal cell, and normality of a cell or a tissue can be determined by identifying the methylation status of a genomic DNA. For example, prediagnosis and diagnosis of diseases such as cancer can be made by determining the methylation state of a genomic DNA (Ushijima 2005, Nat Rev Cancer, 5, 223-31). Furthermore, since a DNA methylation profile consisting of methylation status of numerous T-DMRs over the whole genome is cell-type or tissue-specific, identification of a cell-type or a tissue, differentiation of normal and abnormal cells or tissues, and diagnosis of the presence or absence of a disease can be achieved by using the DNA methylation profile. Furthermore, a T-DMR which is important for diagnosis or identification can be identified.
Methods for investigating the DNA methylation status are classified into methods using selective cleavages with methylation-sensitive restriction enzymes, methods comprising screening for a methylated DNA itself using an antibody or a methylated DNA-binding protein, and methods utilizing a bisulfite reaction.
Examples of methods for obtaining a genome-wide DNA methylation profile includes a restriction landmark genomic scanning (RLGS), which is an analysis using a methylation-sensitive restriction enzyme. In the RLGS, a DNA methylation profile can be visualized as an image composed of many two-dimensional spots by labeling cleavage sites of NotI, a methylation-sensitive restriction enzyme, with a radioactive isotope and performing the cleavage with a non-methylation-sensitive restriction enzyme and two-dimensional electrophoresis in combination (Ohgane, Aikawa et al., 1998, Dev Genet, 22, 132-40). Furthermore, by employing a viRLGS, which simulates the RLGS based on genome database information, in combination, genetic information of spots constituting a DNA methylation profile can also be predicted (Hattori, Abe et al. 2004, Genome Res, 14, 1733-40). To determine whether the predicted genes actually correspond to the spots constituting the profile, however, more detailed analyses are required. In other words, the RLGS is a method suitable for obtaining a genome-wide DNA methylation profile, but the T-DMR identification process is difficult.
Information about recognition sites of specific restriction enzymes can be obtained based on the genome information, and whether these individual regions are methylated can be investigated. In this case, the methylation-sensitive (MS)-PCR method can be used. In the MS-PCR method, PCR primers are designated so that the methylation-sensitive restriction enzyme recognition sites should be flanked, and a fragment treated with a methylation-sensitive restriction enzyme and an untreated fragment are subjected to PCR. Since a fragment is not cleaved when the recognition site of the methylation-sensitive restriction enzyme is methylated, DNA is amplified by PCR. However, since a fragment is cleaved when it is not methylated, amplification by PCR does not occur. Since the amount of the fragments amplified by PCR reflects the amount of non-methylated DNA, the methylation status of a target site can be estimated by quantitating the amount of PCR-amplified fragments. A genome-wide DNA methylation profile can be efficiently obtained by primers at more than one site, when those site have been identified as T-DMRs. Comprehensive analyses of the whole genome based on MS-PCR are enabled by using PCR primer sets established at thousands or tens of thousands of restriction sites (WO2007/088744). However, since which site should be analyzed to identify a new unknown T-DMR is not known, thousands or tens of thousands of reactions need to be performed, which is inefficient.
As comprehensive analysis methods, methods using a microarray have been reported. In the method using a microarray, adjustment of the number of probes to be mounted on the array and fragments to be hybridized is an important factor for determining exhaustiveness. As an analysis method using a methylation-sensitive restriction enzyme, a method of comparing the amounts of fragments produced by methylation-sensitive and non-methylation-sensitive restriction enzymes has been reported. For example, in HpaII tiny fragment enrichment by ligation mediated PCR (HELP) method, fragments treated with a methylation-sensitive enzyme (HpaII) and a non-methylation-sensitive enzyme (MspI) that have an identical recognition sequence are amplified by PCR, and HpaII fragments and MspI fragments produced from the same DNA sample are competitively hybridized with a probe on a microarray to measure the ratio of the amounts of these fragments. This value expresses the methylation status of a specific restriction enzyme recognition sequence (Non-Patent Document 1). Similarly, in Microarray-based integrated analysis of methylation by isoschizomers (MIAMI) method, MspI fragments or HpaII fragments from different samples are competitively hybridized with a probe on a microarray to compare the methylation status of a specific restriction enzyme recognition sequence between different samples (Non-Patent Document 2). To amplify HpaII- and MspI-digested fragments, a ligation-mediated PCR (LM-PCR) method is used in which these fragments are ligated to an adaptor that can be ligated to the cleavage sites and amplified by PCR using a primer specific to the adaptor sequences.
Furthermore, a microarray analysis method is also available utilizing a principle that, when fragments are digested with a non-methylation-sensitive restriction enzyme, then bound with an adaptor, and treated with a methylation-sensitive restriction enzyme, methylated fragments are is amplified, but non-methylated fragments cannot be amplified (same principle as that of MS-PCR). Furthermore, a method, which is claimed to have improved accuracy, is also available by further combining enzyme cleavages intricately.
In general, 2-kb or shorter fragments are efficiently replicated by PCR amplification, and 5-kb or longer fragments are not suitable for amplification. Such a bias in PCR needs to be taken into account in amplification of the above-mentioned non-methylated fragments cleaved with a methylation-sensitive enzyme.
The recognition sequence of HpaII, a methylation-sensitive enzyme, is four base pairs CCGG. When it is assumed that a genomic DNA, in which the four nucleotides are uniformly distributed, is completely digested, fragments of average 265 base pairs are expected. Due to biased distribution of nucleotide sequences are on a mammal genome, however, the frequency of CpG with observed methylation is approx. ⅕ of the expected frequency. Therefore, the frequency of HpaII fragments including CpG is also low. In fact, when an analysis is performed based on the nucleotide sequence of mouse chromosome 17, the average length of HpaII fragments is approx. 1.4 kb, which is longer than when each nucleotide is assumed to be uniformly distributed. Due to methodological restrictions of PCR and the positional bias of recognition sites of restriction enzymes, the following problems arise in analyses using a single recognition site.
For example, when an analysis is performed using cleavage of a fragment with HpaII as an indicator, three HpaII sites A, B, and C shown in
In other words, when a restriction enzyme with a long recognition sequence is used, most fragments are too long to be amplified by PCR. For example, NotI used in the RLGS recognizes and cleaves an 8-bp sequence, GCGGCCGC. Since the average chain length of the fragment expected from the mouse genome sequence is 371 kb, most fragments cannot be amplified by PCR (details are described in Reference Example). The SmaI recognition sequence is 7 kb in length, and most fragments are not to be analyzed when the LM-PCR method is used. Furthermore, since the occurrence of amplification of a fragment depends on the methylation state of one of the two ends as shown using HpaII as an example, the analysis will become less comprehensive.
As described above, since a digested fragment is not amplified by LM-PCR when the distance between cleavage sites of a methylation-sensitive restriction enzyme is long, there is a problem that the methylation status of a cleavage site cannot be determined. Against such a technical background, an object of the present invention is to provide means with which the occurrence of a cleaved fragment can be correctly determined even when the distance between cleavage sites of a methylation-sensitive restriction enzyme is long.
The inventors of the present invention conducted various researches to achieve the foregoing object. As a result, they found that the methylation status of a cleavage site can be correctly determined even when the distance between cleavage sites of a methylation-sensitive restriction enzyme is long, by treating a genomic DNA with a methylation-sensitive restriction enzyme and then treating with a non-methylation-sensitive restriction enzyme to selectively amplify only a fragment having cleavage ends of the methylation-sensitive restriction enzyme and the non-methylation-sensitive restriction enzyme, and accomplished the present invention.
Specifically, the present invention provides the following [1] to [8].
[1] A method for amplifying a DNA fragment, characterized by comprising the following steps (1) to (5):
(1) digesting a sample double-stranded DNA with a first restriction enzyme;
(2) adding a double-stranded DNA fragment consisting of a first oligonucleotide and a second oligonucleotide having a sequence complementary to the first oligonucleotide to a restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 5′ end of the first oligonucleotide and the 3′ end of the second oligonucleotide are ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1);
(3) digesting the DNA fragment obtained in step (2) with a second restriction enzyme;
(4) allowing the double-stranded DNA fragment produced in step (3) to coexist with a third oligonucleotide with an unphosphorylated 5′ end and a fourth oligonucleotide with an unphosphorylated 5′ end that has a sequence complementary to the third oligonucleotide in the presence of a ligase, thereby adding a double-stranded DNA fragment consisting of the third oligonucleotide and the fourth oligonucleotide to the second restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3) to obtain a DNA fragment, in which the 3′ end of the fourth oligonucleotide is ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3), but the 5′ end of the third oligonucleotide is not ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3); and
(5) heating the double-stranded DNA fragment obtained in step (4) thereby dissociating the double-stranded DNA fragment, and then performing a polymerase chain reaction using an oligonucleotide hybridizable with the first oligonucleotide and an oligonucleotide hybridizable with the third oligonucleotide as primers to selectively amplify a DNA fragment having the cleavage end of the first restriction enzyme and the cleavage end of the second restriction enzyme.
[2] The method for amplifying a DNA fragment according to [1], characterized in that the step (2) is allowing the double-stranded DNA fragment produced in step (1) to coexist with the first oligonucleotide with an unphosphorylated 5′ end and the second oligonucleotide with an unphosphorylated 5′ end in the presence of a ligase, thereby adding a double-stranded DNA fragment consisting of the first oligonucleotide and the second oligonucleotide to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 3′ end of the second oligonucleotide is ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), but the 5′ end of the first oligonucleotide is not ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), and then allowing a DNA polymerase to act to ligate the double-stranded DNA fragment produced in step (1) and the 5′ end of the first oligonucleotide.
[3] The method for amplifying a DNA fragment according to [1], characterized in that the step (2) is allowing the double-stranded DNA fragment produced in step (1) to coexist with the first oligonucleotide with a phosphorylated 5′ end and the second oligonucleotide with a phosphorylated 5′ end in the presence of a ligase, thereby adding a double-stranded DNA fragment consisting of the first oligonucleotide and the second oligonucleotide to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 5′ end of the first oligonucleotide and the 3′ end of the second oligonucleotide are ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), and then removing the first oligonucleotide, the second oligonucleotide, and the DNA fragment produced by ligation.
[4] The method for amplifying a DNA fragment according to any of [1] to [3], characterized in that the first restriction enzyme and the second restriction enzyme are restriction enzymes which leave a 5′ overhang, and the first oligonucleotide and the third oligonucleotide are oligonucleotides having a sequences complementary to the 5′ overhangs generated by the first restriction enzyme and the second restriction enzyme, respectively.
[5] A method for measuring the methylation levels of a genomic DNA by allowing a methylation-sensitive restriction enzyme to act on the genomic DNA, amplifying the resulting DNA fragment, and determining the methylation degree of a cleavage site of the methylation-sensitive restriction enzyme based on the amplification degree, characterized by comprising the following steps (1) to (6):
(1) digesting a genomic DNA with a methylation-sensitive restriction enzyme;
(2) adding a double-stranded DNA fragment consisting of a first oligonucleotide and a second oligonucleotide having a sequence complementary to the first oligonucleotide to a restriction enzyme cleavage end of a DNA fragment produced in step (1) to obtain a DNA fragment, in which the 5′ end of the first oligonucleotide and the 3′ end of the second oligonucleotide are ligated to the restriction enzyme cleavage end of the DNA fragment produced in step (1);
(3) digesting the DNA fragment obtained in step (2) with a non-methylation-sensitive restriction enzyme;
(4) allowing the double-stranded DNA fragment produced in step (3) to coexist with a third oligonucleotide with an unphosphorylated 5′ end and a fourth oligonucleotide with an unphosphorylated 5′ end that has a sequence complementary to the third oligonucleotide in the presence of a ligase, thereby adding a double-stranded DNA fragment consisting of the third oligonucleotide and the fourth oligonucleotide to a non-methylation-sensitive restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3) to obtain a DNA fragment, in which the 3′ end of the fourth oligonucleotide is ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3), but the 5′ end of the third oligonucleotide is not ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3);
(5) heating the double-stranded DNA fragment obtained in step (4) thereby dissociating the double-stranded DNA fragment, and then performing a polymerase chain reaction using an oligonucleotide hybridizable with the first oligonucleotide and an oligonucleotide hybridizable with the third oligonucleotide as primers to selectively amplify a DNA fragment having a cleavage end of a methylation-sensitive restriction enzyme and a cleavage end of a non-methylation-sensitive restriction enzyme; and
(6) identifying the position of the amplified DNA fragment on the genomic DNA and determining the amplification amount of a DNA fragment including a cleavage site for each methylation-sensitive restriction enzyme.
[6] The method for measuring the methylation degree of a genomic DNA according to [5], characterized in that the step (2) is allowing the double-stranded DNA fragment produced in step (1) to coexist with a first oligonucleotide with an unphosphorylated 5′ end and a second oligonucleotide with an unphosphorylated 5′ end in the presence of a ligase, and adding a double-stranded DNA fragment consisting of the third oligonucleotide and the fourth oligonucleotide to a restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 3′ end of the second oligonucleotide is ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), but the 5′ end of the first oligonucleotide is not ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), and then allowing a DNA polymerase to act to ligate the double-stranded DNA fragment produced in step (1) and the 5′ end of the first oligonucleotide.
[7] The method for measuring the methylation degree of a genomic DNA according to [5], characterized in that the step (2) is allowing the double-stranded DNA fragment produced in step (1) to coexist with a first oligonucleotide with a phosphorylated 5′ end and a second oligonucleotide with a phosphorylated 5′ end in the presence of a ligase, thereby adding a double-stranded DNA fragment consisting of the first oligonucleotide and the second oligonucleotide to a restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 5′ end of the first oligonucleotide and the 3′ end of the second oligonucleotide are ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), and removing the first oligonucleotide, the second oligonucleotide, and the DNA fragment produced by ligation.
[8] The method for measuring the methylation degree of a genomic DNA according to any one of [5] to [7], characterized in that the methylation-sensitive restriction enzyme and the non-methylation-sensitive restriction enzyme are restriction enzymes that leave a 5′ overhang, and the first oligonucleotide and the third oligonucleotide are oligonucleotides having sequences complementary to the 5′ overhangs generated by the methylation-sensitive restriction enzyme and the non-methylation-sensitive restriction enzyme, respectively.
According to the method for amplifying a DNA fragment of the present invention, whether a DNA has been cleaved with a restriction enzyme can be correctly determined. By using this amplification method for an analysis of the methylation status of a genomic DNA, the methylation levels of the genomic DNA can be measured simply and correctly as well as comprehensively. Furthermore, the amplification method of the present invention can also be utilized in not only analyses of methylation status but also analyses of single nucleotide polymorphism.
Hereafter, the present invention will be explained in detail.
First, the principle of the method for amplifying a DNA fragment of the present invention will be explained with reference to
A genomic DNA (
After the above procedures, the DNA fragment is cleaved with a second restriction enzyme, and the cleaved fragment is allowed to coexist with a third oligonucleotide 3 and a fourth oligonucleotide 4 in the presence of a ligase. Since the 5′ end of the third oligonucleotide 3 is not phosphorylated as with the first oligonucleotide 1, this end is not ligated to the 3′ end of a fragment cleaved with the second restriction enzyme (
Each step of the method for amplifying a DNA fragment of the present invention will be explained below.
Step (1) is digesting a sample double-stranded DNA with the first restriction enzyme. The first restriction enzyme is not limited, but, since the method for amplifying a DNA fragment of the present invention is mainly used in the analysis of the methylation state of a genomic DNA, methylation-sensitive restriction enzymes are preferred. Examples of the methylation-sensitive restriction enzyme include AatII, AclI, AgeI, AscI, AvaI, BmgBI, BsiWI, BspDI, BstBI, ClaI, EagI, FseI, FspI, HaeII, HhaI, Hpy99I, HpyCH4IV, HpaII, KasI, MluI, NarI, NaeI, NotI, NruI, PmlI, PvuI, RsrII, SacII, SalI, SfoI, SgrAi, SmaI, SnaBI, and so forth. Since the method for amplifying a DNA fragment of the present invention can also be used for analyses of single nucleotide polymorphism or the like, a restriction enzyme that cleaves only a specific genotype DNA or the like may be used as the first restriction enzyme. Furthermore, the first restriction enzyme may produce any of a blunt-end, a 5′ overhang, and a 3′ overhang. The sample double-stranded DNA is not particularly limited, and a genomic DNA or a DNA of a gene having polymorphism can be used as a sample. A problem may occur in procedures such as DNA extraction when a DNA fragment produced by the first restriction enzyme is very long. Therefore, in this case, a sample double-stranded DNA may be cleaved with another restriction enzyme other than the first restriction enzyme and the second restriction enzyme.
Step (2) is adding a double-stranded DNA fragment consisting of the first oligonucleotide and the second oligonucleotide having a sequence complementary to the first oligonucleotide to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 5′ end of the first oligonucleotide and the 3′ end of the second oligonucleotide are ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1). The first oligonucleotide and the second oligonucleotide may be similar to oligonucleotides referred to as adaptors in usual LM-PCR. The length of the first oligonucleotide is not particularly limited and is preferably 6 to 50 nucleotides. The length of the second oligonucleotide is not particularly limited either and is preferably 6 to 50 nucleotides. In order to add a double-stranded DNA fragment consisting of the first oligonucleotide and the second oligonucleotide to the double-stranded DNA fragment produced in step (1), it is necessary that the double-stranded DNA fragment produced in step (1) is allowed to coexist with the first oligonucleotide with an unphosphorylated 5′ end and the second oligonucleotide with an unphosphorylated 5′ end in the presence of a ligase, thereby adding the double-stranded DNA fragment consisting of the first oligonucleotide and the second oligonucleotide to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1) to obtain a DNA fragment, in which the 3′ end of the second oligonucleotide is ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), but the 5′ end of the first oligonucleotide is not ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (1), and then a DNA polymerase is allowed to act to ligate the double-stranded DNA fragment produced in step (1) and the 5′ end of the first oligonucleotide. It is sufficient to use a ligase used in usual LM-PCR, and T4 ligase, E. coli DNA ligase, Tag DNAl ligase, or the like can be used, for example. As a DNA polymerase, a DNA polymerase I Klenow fragment can be used. Enzymes other than Klenow fragments may be used as a DNA polymerase, but enzymes with a weak 5′→3′ exonuclease activity are preferred. Examples of such enzymes include Sequenase, recombinant Taq DNA polymerase, and similar enzymes whose exonuclease activity has been eliminated. Even if enzymes have an exonuclease activity, there is no problem when the enzymes are reacted under conditions under which the exonuclease activity is weak. Therefore, T4 DNA polymerase, DNA polymerase I, natural Taq DNA polymerase, and similar enzymes may be used. Furthermore, instead of an oligonucleotide with an unphosphorylated 5′ end, a phosphorylated oligonucleotide may be used. In this case, treatment with a DNA polymerase can be omitted. However, since the oligonucleotides may be ligated to each other, resulting in competitive inhibition of subsequent PCR reactions, procedures for removing low molecular weight DNA, such as gel filtration, spin column, ethanol precipitation, isopropanol precipitation, and polyethylene glycol precipitation, are preferably performed. In step (5) described later, a polymerase chain reaction occurs by binding of the second oligonucleotide or the like to the sequence of the first oligonucleotide. Therefore, the first oligonucleotide preferably has a sufficient length that enables hybridization with the whole second oligonucleotide. However, when treatment with a DNA polymerase is performed, the first oligonucleotide may be a short oligonucleotide that can be hybridized with only a part of the second oligonucleotide. In this case, since a complementary strand of the second oligonucleotide is synthesized during the process in which a DNA polymerase repairs a gap between the first oligonucleotide and the first enzyme cleavage end, a portion corresponding to the first oligonucleotide has a sequence hybridizable with the whole second oligonucleotide as a result.
Step (3) is digesting the DNA fragment obtained in step (2) with the second restriction enzyme. The second restriction enzyme is not particularly limited, and enzymes that produce a short cleavage fragment are preferred. Preferred examples include enzymes recognizing four nucleotides such as TaqI. Furthermore, when the methylation state of a genomic DNA is analyzed, a non-methylation-sensitive restriction enzyme is preferably used as the second restriction enzyme. The second restriction enzyme may also be an enzyme that produces any kind of end, as with the first restriction enzyme.
Step (4) is allowing the double-stranded DNA fragment produced in step (3) to coexist with the third oligonucleotide with an unphosphorylated 5′ end and the fourth oligonucleotide with an unphosphorylated 5′ end having a sequence complementary to the third oligonucleotide in the presence of a ligase, thereby adding a double-stranded DNA fragment consisting of the third oligonucleotide and the fourth oligonucleotide to the second restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3) to obtain a DNA fragment, in which the 3′ end of the fourth oligonucleotide is ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3), but the 5′ end of the third oligonucleotide is not ligated to the restriction enzyme cleavage end of the double-stranded DNA fragment produced in step (3). Ligases similar to the ligases explained in step (2) can be used. Furthermore, oligonucleotides similar to the first oligonucleotide and the second oligonucleotide explained in step (2) can also be used as the third oligonucleotide and the fourth oligonucleotide. However, when the sequences are identical or similar, such a problem may arise that the primer to be hybridized with the first oligonucleotide in step (5) may be hybridized with the third oligonucleotide. Therefore, oligonucleotide sequences that are not similar should be used. Furthermore, since the third oligonucleotide-dependent PCR reaction may produce nonspecific reaction products, it is preferable that oligonucleotides should not be hybridized with the fourth oligonucleotide or the like under PCR reaction conditions.
Step (5) is heating the double-stranded DNA fragment obtained in step (4) thereby dissociating the double-stranded DNA fragment, and then performing a polymerase chain reaction using an oligonucleotide hybridizable with the first oligonucleotide and oligonucleotide hybridizable with the third oligonucleotide as primers to selectively amplify a DNA fragment having the cleavage end of the first restriction enzyme and the cleavage end of the second restriction enzyme. The temperature at the time of heating can be similar to temperature in usual hot start PCR, and appropriate temperature is approx. 90 to 95° C. The polymerase chain reaction conditions (temperature, number of cycles, polymerase used, etc.) can also be similar to those in usual hot start PCR. Examples of the oligonucleotide hybridizable with the first oligonucleotide used as a primer include the second oligonucleotide, an oligonucleotide obtained by removing one or several nucleotides of the second oligonucleotide, an oligonucleotide obtained by adding another sequence to the above-mentioned oligonucleotides to an extent that the function as a primer should not be affected, and so forth. Furthermore, examples of the oligonucleotide hybridizable with the third oligonucleotide include the fourth oligonucleotide, an oligonucleotide obtained by removing one or several nucleotides of the fourth oligonucleotide, an oligonucleotide obtained by adding another sequence to the above-mentioned oligonucleotides to an extent that the function as a primer should not be affected, and so forth. The length of these oligonucleotides used as primers is not particularly limited and is preferably 17 to 50 nucleotides.
The method for amplifying a DNA fragment of the present invention can be used to measure the methylation degree of a genomic DNA. Specifically, using a genomic DNA as a sample double-stranded DNA, a methylation-sensitive restriction enzyme as the first restriction enzyme, and a non-methylation-sensitive restriction enzyme as the second restriction enzyme, the position of a DNA fragment amplified on the genomic DNA is identified, the amplification amount of a DNA fragment including the cleavage site is determined for each methylation-sensitive restriction enzyme cleavage site, and the methylation degree of the methylation-sensitive restriction enzyme cleavage site can be measured. For example, when a small amount of DNA fragments including a certain methylation-sensitive restriction enzyme cleavage site are amplified, the cleavage site can be determined to be in a hypermethylated state. On the contrary, when a large amount of DNA fragments are amplified, the cleavage site can be determined to be in a hypomethylated state. The method for identifying the position of the amplified DNA fragment on the genome is not particularly limited, and a genome tiling array or the like can be used.
The method for amplifying a DNA fragment of the present invention can be used for analyses of single nucleotide polymorphism, detection or analyses of recombination of DNA including transposition, deletion, translocation, and the like, and point or region mutation, and the like in addition to measurement of the methylation degree of the genomic DNA.
Hereafter, the present invention will be explained more specifically with the following examples.
Restriction enzyme sites of a mouse genome sequence (mm6) obtained from the UCSC Genome Browser Database (http://genome.ucsc.edu/) were analyzed with a sequence analysis software (EMBOSS Package), and detailed analyses were further performed by using the obtained results in software programs such as Excel, File Maker, Perl, R, and Python.
For example, 6012 NotI recognition sites exist in the mouse genome sequence (of the genomic sequences, ChrM, a random sequence of chromosome, is not included). In a histogram showing a distribution of lengths of these fragments (
On the other hand, in a histogram similarly showing a distribution of chain lengths of fragments cleaved with NotI and TaqI (
Meanwhile, approx. 200 times more TaqI recognition sites than NotI sites exist, and the average chain length of fragments are approx. 1.8 kb. Therefore, most fragments cleaved with TaqI are in the fragment length range in which fragments are very likely to be amplified by PCR. It is found that, if a fragment having only the TaqI cleavage end is similarly amplified as with the fragment having the NotI cleavage end, most amplified fragments are fragments having only the TaqI cleavage end at an end thereof.
The restriction sites of HpyCH4IV were similarly analyzed. The mouse genome has approx. 3.5 million HpyCH4IV recognition sites (of the genomic sequences, ChrM, a random sequence of chromosome, is not included). As a representative, an example of an analysis using the sequence of mouse chromosome 17 will be described. Chromosome 17 has 64,250 HpyCH4IV recognition sites. In a histogram showing a distribution of fragment lengths (
As in the case of NotI, in a histogram showing a distribution of chain lengths of fragments cleaved with HpyCH4IV and TaqI (
The genomic DNA of a target cell was prepared as follows by a known method (Molecular Cloning; A Laboratory Manual). Each frozen cell sample (approx. 1 or 2×106 cells) was dissolved in 100 μl of lysis buffer [containing 5 mM EDTA, 100 mM Tris-HCl [pH 8.5], 0.2% SDS, proteinase K (200 μg/ml)], and the mixture was incubated at 55° C. for 30 min. The mixture was extracted twice with phenol/chloroform/isoamyl alcohol (50:49:1), and the genomic DNA was precipitated by ethanol precipitation and dissolved in 20 μl of TE (10 mM Tris-HCl, 1 mM EDTA [pH 7.6]).
A methylation-sensitive restriction enzyme NotI (Takara) and a non-methylation-sensitive restriction enzyme PvuII (Takara) were added to 5 μg of the genomic DNA diluted in a restriction enzyme buffer and reacted overnight at 37° C. to digest the genomic DNA. The mixture was extracted with phenol/chloroform (1:1) to recover an aqueous layer containing DNA, followed by the addition of 1/10 of the volume of 3 M sodium acetate solution (pH 5.2), and then the genomic DNA was precipitated by ethanol precipitation. The recovered DNA was dissolved in 5 μl of TE solution, and 1/10 of the volume thereof was subjected to agarose electrophoresis to confirm the cleavage of DNA fragments. 1 pmol of adaptors (R24 and Rggcc12) were added to 1/10 of the volume of DNA, followed by the addition of a T4 ligase buffer, and then the mixture was heated at 65° C. for 5 min and gradually cooled to 16° C. over approx. 1 h. T4 DNA ligase (NEB) was added here and reacted overnight at 16° C. to ligate the genomic DNA cleaved with the restriction enzyme and the adaptors. Of the adaptors used, R24 has a sequence complementary to Rggcc12, and Rggcc12 has a sequence complementary to the overhang portion produced by NotI. After the overnight reaction, a dNTP mixture solution and a Klenow fragment (Takara) were added, and a reaction was further performed at 16° C. for 30 min. The reaction mixture was heated at 70° C. for 10 min to inactivate the DNA ligase and the Klenow fragment. A sodium chloride solution was added here at a final concentration of 100 mM, followed by the addition of restriction enzyme TaqI, and the mixture was reacted at 65° C. for 3 h to completely cleave the DNA. The mixture was extracted with phenol/chloroform (1:1) to recover an aqueous layer containing DNA, unreacted adaptors were removed using MicroSpin Column (GE Healthcare), followed by the addition of 1/10 of the volume of 3 M sodium acetate solution (pH 5.2), and DNA was precipitated by ethanol precipitation. DNA was dissolved in an appropriate amount of sterilized water, followed by the addition of 100 pmol of adaptors (addition of equimolar amounts of N24 and Ncg10) and a T4 ligase buffer, and then the mixture was heated at 65° C. for 5 min and then gradually cooled to 16° C. over approx. 1 h. T4 DNA ligase (NEB) was added here, and the mixture was reacted overnight at 16° C. to ligate the DNA cleaved with restriction enzyme TaqI and the adaptors. Of the adaptors used, N24 has a sequence complementary to Ncg10, and Ncg10 has a sequence complementary to the overhang portion produced by TaqI. After completion of the reaction, DNA fragments were purified using a DNA purification kit (Wizard SV Gel and PCR Clean-Up System, Promega) to remove unreacted adaptors and the like. A part of the recovered DNA was added to a modified heat-resistant DNA polymerase reaction mixture containing primers N18 and R182 and treated in a reaction mixture containing dNTP at 95° C. for 7 min, ant then a reaction was performed 20 cycles consisting of heating cycles at 94° C. for 30 s, 62° C. for 30 s, and 72° C. for 2 min. Of the primers used, N18 has a sequence obtained by removing one to several nucleotides from an end of N24, and R182 has a sequence obtained by removing one to several nucleotides from an end of R24. As controls, reaction mixtures containing N18 alone or R182 alone as a primer were prepared and similarly reacted. In the controls, when DNA is cleaved with TaqI alone, fragments can be amplified, and all genome sequences having a methylated NotI cleavage site are amplified.
According to the principle of usual LM-PCR, fragments having only the NotI cleavage end at both ends are amplified in the PCR reaction mixture containing R182 alone, fragments having only the TaqI cleavage end at both ends are amplified in the PCR reaction mixture containing N18 alone, and fragments having NotI and TaqI cleavage ends are amplified in the reaction mixture containing N18 and R182 in addition to the above-mentioned two kinds of fragments.
Before amplification, fragments having only the TaqI cleavage end at both ends are most abundant. Even if it is assumed that all NotI sites in the genome are non-methylated, approx. 250 times more fragments having only the TaqI cleavage end at both ends exist than fragments having NotI-TaqI ends. Therefore, when usual LM-PCR is performed, fragments amplified with N18 will be the most abundant. As shown in
After completion of the reaction, the PCR products in the reaction mixture containing N18 and R182 were purified using QIAquick Mini Elute Kit. Furthermore, for comparison, usual LM-PCR was performed using N18 as a primer, and the PCR products were purified using QIAquick Mini Elute Kit.
A total of 9 μg each of the above-mentioned two kinds of purified PCR products were sent to NimbleGen, and the products of usual LM-PCR using N18 as a primer were labeled with Cy5, the products amplified by the method of the present invention using both N18 and R182 as primers were labeled with Cy3, and these products were hybridized with a tiling array containing sequences surrounding the NotI sequence in a competitive hybridization system to obtain signals. Here, the sequences surrounding NotI means a region including 2.5 kb upstream and 2.5 kb downstream of NotI based on the information obtained by extracting the NotI restriction site from a mouse genome sequence (mm7) obtained from the UCSC Genome Browser Database (http://genome.ucsc.edu/) using EMBOSS (Trends in Genetics 16, 276-277). A probe sequence was selected so that one 50 nucleotide-long probe should be included per 100 base pairs in these regions. The probe corresponding to the repeat sequence (Repeat Mask) was removed.
Of the analysis files provided by NimbleGen, data with normalized signals using the q-spline function were analyzed. The obtained results were further analyzed in detail using software programs such as Excel, File Maker, Perl, R, and Python.
It was assumed that all probes on the microarray were completely cleaved, and probes that were longer than 50 by and 5 kb and shorter, were classified into probes existing in a NotI-TaqI fragment or a NotI-NotI fragment and other probes (mainly probes existing in a TaqI-TaqI fragment) (hereinafter, the former is referred to as “NotI-TaqI group” and the latter as “TaqI-TaqI group”).
Logarithmic values of the fluorescence intensity ratio of Cy3 and Cy5 of the probes belonging to the NotI-TaqI group (Cy5/Cy3) were obtained and plotted in a histogram (
Meanwhile, a histogram was similarly prepared for the probes belonging to the TaqI-TaqI group (
The signals for hybridization of amplification products by the method of the present invention and probes on the microarray were expressed using Integrated Genome Browser (IGB, Affymetrix, Inc.).
As illustrated in
As shown in
A methylation-sensitive restriction enzyme HpyCH4IV (NEB) was added to 5 μg of a genomic DNA diluted in a restriction enzyme buffer and reacted overnight at 37° C. to digest the genomic DNA. The mixture was extracted with phenol/chloroform (1:1) to recover an aqueous layer containing DNA, followed by the addition of 1/10 of the volume of 3 M sodium acetate solution (pH 5.2), and then the genomic DNA was precipitated by ethanol precipitation. The recovered DNA was dissolved in 5 μl of TE solution, and 1/10 of the volume of the mixture was subjected to agarose electrophoresis to confirm the cleavage of DNA fragments. 100 pmol of adaptors (R24 and R10) were added to 1/10 of the volume of DNA, followed by the addition of a T4 ligase buffer, and the mixture was heated at 65° C. for 5 min and then gradually cooled to 16° C. over approx. 1 h. T4 DNA ligase (NEB) was added here, and this mixture was reacted overnight at 37° C. to ligate the genomic DNA cleaved with the restriction enzyme and the adaptors. Of the adaptors used, R10 has a sequence complementary to the HpyCH4IV cleavage end. After overnight reaction, a dNTP mixture solution and a Klenow fragment (Takara) were added, and a reaction was further performed at 16° C. for 30 min. The reaction mixture was heated at 70° C. for 10 min to inactivate the DNA ligase and the Klenow fragment. A sodium chloride solution was added here at a final concentration of 100 mM, followed by the addition of restriction enzyme TaqI, and the mixture was reacted at 65° C. for 3 h to completely cleave the DNA. The mixture was extracted with phenol/chloroform (1:1) to recover an aqueous layer containing DNA, followed by the addition of 1/10 of the volume of 3 M sodium acetate solution (pH 5.2), and DNA was precipitated by ethanol precipitation. DNA was dissolved in an appropriate amount of sterilized water, followed by the addition of 100 pmol of adaptors (addition of equimolar amounts of N24 and Ncg10), followed by the addition of a T4 ligase buffer, and the mixture was heated at 65° C. for 5 min and then gradually cooled to 16° C. over approx. 1 h. T4 DNA ligase (NEB) was added here, and the mixture was reacted overnight at 16° C. to ligate the DNA cleaved with restriction enzyme TaqI and the adaptors. After completion of the reaction, DNA fragments were purified using a DNA purification kit (Wizard SV Gel and PCR Clean-Up System, Promega) to remove unreacted adaptors and the like. A part of the recovered DNA was added to a modified heat-resistant DNA polymerase reaction mixture containing primers N18 and R182 and treated in a reaction mixture containing dNTP and dUTP at 95° C. for 7 min, and a reaction was performed 20 cycles consisting of thermal cycles at 94° C. for 30 s, 62° C. for 30 s, and 72° C. for 2 min. As controls, reaction mixtures containing primer N18 alone or R182 alone were prepared and subjected to similar reactions. As shown in
A total of 9 μg of PCR products amplified using primers N18 and R182 were fragmented using Gene Chip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) according to a method recommended by the manufacturer and labeled with biotin. According to a method recommended by the manufacturer, the products were hybridized with a mouse promoter-tiling array to obtain hybridization signals. Signals were analyzed by GCOS to obtain files (.CEL) in which results were compiled as data.
For analysis of the .CEL file, R and MAT were used (Proc Natl Acad Sci USA. 103: 12457-12462). Using the same liver DNA, preparation of fragments and hybridization with the microarray described in Example 5 were performed twice. Correlations of the signal intensity on the microarray were plotted to obtain the correlation coefficient (
By performing analyses using MAT, the hybridization signal values are converted to MATscores, to which effect of probe sequences on signal intensity and redundancy of a probe is added, and can be expressed with IGB. Results of MAT scores of regions of which methylation state had already been found by IGB are shown in
Furthermore, the region with a high MAT score was centered around the HpyCH4IV cleavage site and overlapped a region surrounded by the HpyCH4IV cleavage site and the TaqI cleavage site. Therefore, it was confirmed that HpyCH4IV-TaqI fragments had been selectively amplified by the amplification method of the present invention.
When distributions of MATscores were compared between the probes existing in a fragment including the HpyCH4IV cleavage site and the other probes, the probes existing in a fragment including the HpyCH4IV cleavage site showed a distribution of high scores as shown in
The sequences of the adaptors and primers used in this example are listed below.
This specification encompasses contents described in the specification and/or drawings of Japanese Patent Application No. 2007-056839, based on which the conventional priority of the present application was claimed. Furthermore, various publications, patents, and patent applications referred to throughout the present invention in their entireties are hereby incorporated by reference into this specification.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-056839 | Mar 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/053941 | 3/5/2008 | WO | 00 | 9/4/2009 |
