WO2007106802A2

WO2007106802A2 - Method for linear amplification of bisulfite converted dna

Info

Publication number: WO2007106802A2
Application number: PCT/US2007/063860
Authority: WO
Inventors: Zheng Li
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2006-03-14
Filing date: 2007-03-13
Publication date: 2007-09-20
Also published as: WO2007106802A8; WO2007106802A3

Abstract

Provided is a method of detecting DNA methylation at CpG sites and at SNP sites in a sample through the linear amplification of bisulfite converted DNA (referred to as the 'L-ABCD' method). Unlike prior art DNA methylation assays, the L-ABCD method of the present invention may be applied on single-stranded DNA and from samples that have degraded DNA. The amplification products of the L-ABCD method may be assayed by using methods known in the art such as methylation specific primer extension ('MSPE'), DNA microarrays, microbead assays, and planar waveguid ('PWG') chips.

Description

METHOD FOR LINEAR AMPLIFICATION OF BISULFITE CONVERTED DNA

BACKGROUND or 11 π. INVΓN \ ION

Within the human and animal genome, sites where cytosine is located 5' of guanine are referred to as CpG sites. CpG sites are sites responsible for the only known natural modification of DNA in humans and animals, which is DNA methylation. At CpG sites, DNA methylation occurs when DNA (cytosine-5) methyltransferase ("DNA-mtase") catalyzes the methylation by adding a methyl group from S-adenosyl-L-methionine to the fifth carbon position of the cytosine. Seventy to eighty percent of human CpG sites are methylated; however, the methylation occurs in sites of low density. By contrast, in CpG islands, i.e., sites of high CpG density, most of the CpG sites are uninethylated.

DNA methylation at CpG sites is propagated through cell division and mediates epigenetic inheritance, i.e., changes in gene expression not associated with DNA sequence changes. DNA methylation known to play a role in regulating gene expression during development and is associated with transcriptional silencing of imprinted genes and the silencing of the heavily methylated X chromosome in females. DNA methylation of normally unmethylated cytosines in CpG islands has been implicated in genomic imprinting disorders, where only one allele of a parent is expressed while the other is not, as well as the transcriptional silencing of tumor suppressor genes, which may lead to the growth of cancerous tumors.

In addition to the foregoing, changes at CpG sites (not necessarily at CpG islands) are the most frequent source of human genetic variation and such changes have been found to be responsible for a significant number of single nucleotide polymorphisms ("SNPs^'') in mammalian genomes. See, Daly et al., NAl URC GbNEl ICS 29:229-232 (2001 ), Nachman et al., GΓNΓ [ ICS 156, 297-304 (2000), and Krawczak et al., AM J HUM GLNΓ I 63, 474-488 ( 1998).

CpG island methylation is identified using methylation-sensitive restriction enzymes or by distinguishing methylated from unmethylated DNA through bisulfite conversion of DNA, which when completed, converts unmethylated cytosine to uracil, but leaves methylated cytosine intact. Identification of CpG islands through both methylation-sensitive restriction enzymes and bisulfite conversion of DNA require an amplification step, such as DNA hybridization (i.e., Southern analysis), polymerase chain reaction ("PCR^*'), or ligase chain reaction ("LCR") in order to visualize the DNA.

A problem inherent with the use of PCR to amplify bisulfite converted DNA is PCR bias, which occurs when methylated and unmethylated DNA molecules amplify with greatly differing efficiencies. In order to avoid the sequence-dependent and length-dependent biases inherent with the exponential amplification that characterizes the PCR assay, researchers have developed various isothermal amplification techniques. In the isothermal technique known as helicase-dependent isothermal DNA amplification ("ΗDA^"'), DNA helicase separates double-stranded DNA ("dsDNA^"') and generates single-stranded templates for primer hybridization and subsequent extension. Vincent et at., EMBO REPORTS 5(8):795-800 (2004). HDA has the disadvantage of requiring the design of target-specific primers.

In the isothermal technique known as multiple displacement amplification ("MDA^") dsDNA is amplified using two primers that are complementary to the two individual strands of DNA, respectively, with amplification proceeding by replication initiated at each primer through the sequence of interest with intervening primers becoming displaced through the use of the a polymerase, such as phi29 polymerase. U.S. Patent No. 6, 124, 120 to Lizardi. A disadvantage of MDA is that it requires high molecular weight DNA in order to run the assay.

In 1996, Phillips and Eberwine described an antisense RNA ("aRNA") amplification method to linearly amplify mRNA from living cells. Phillips and Eberwine, ME I ΠODS : A COMPANION I O ME THODS IN ENZYMOLOGY I O (283-288 1996). The aRNA method entailed tagging living cells with poty(T) tails such that the cDNA transcribed thereon contained T7 promoter sequences. After processing a second strand of cDNA synthesis, T7 polymerase was used for amplification. In 2003, Liu et al. used the method first described by Phillips and Eberwine to amplify genomic DNA by adding a polyA tail to the ends of DNA fragments and processing a second strand of DNA using a 11- polyA primer to form double-stranded templates suitable for in vitro transcription. Liu et al., BMC GENOMICS 4( 19) (2003) (open access at www.biomedicalcentral.com/1471 -2164/4/19). Neither Phillips and Eberwine nor Liu et al. developed the linear amplification assay for application to single stranded DNA.

In order to determine if methylated CpG islands are involved in epigenetic disorders, a reliable technique for identifying and quantifying methylated DNA is imperative. As noted above, the DNA methylation assays currently in the art all have disadvantages that compromise the efficiency or reliability of the assays for the identification and quantitation of DNA methylation in CpG islands.

Another problem that is prevalent in the conventional DNA methylation assays used in the art is that the bisulfite used to convert the DNA has the disadvantage of degrading the DNA thus making the majority of the DNA in the sample useless for the conventional assays; even where some DNA is not degraded, the quantity of non-degraded DNA remaining in the sample after the bisulfite conversion is generally so small that only a few assays are possible and consequently, the interrogation of many targets is impossible. Thus, there also is a need in the art for a method of preserving DNA in a sample that is undergoing bisulfite conversion and/or a method of amplifying degraded DNA in a sample. SUMMARY OF TI »-; INVENTION

The present invention overcomes the shortcomings of the DNA methylation assays known in the art by providing a reliable and accurate method for preserving, linearly amplifying, and labeling bisulfite treated, single stranded DNA that does not require any of the following: a PCR step, target specific primers, or high molecular weight DNA. The method of the present invention, referred to herein as the "L-ABCD" method (L-ABCD being short for linear amplification of bisulfite converted DNA) has the surprising and unexpected result of being capable of amplifying DNA from very small samples having degraded DNA.

In one aspect of the invention, there is provided a method of linearly amplifying bisulfite converted DNA comprising the steps of: (a) obtaining DNA from a sample to be analyzed and where appropriate denaturing the DNA to form single-stranded DNA; (b) generating modified DNA by reacting the single-stranded DNA with an agent that converts unmethyiated cytosiπe residues at CpG sites on the DNA to uracil while leaving CpG sites with methylated cytosine residues unchanged; (c) treating the modified DNA with an enzyme that releases phosphate groups from 3' ends of the modified DNA; (d) adding a homopolymer tail to the 3' end of the modified DNA by reacting the modified DNA with single nucleotide dNTPs in the presence of an enzyme; (e) annealing a primer to the homopolymer tail of the modified DNA, wherein the primer has a homopolymer tail that is complementary to the homopolymer tail of the modified DNA; (f) initiating DNA replication by reacting the modified DNA of step (e) with dNTPs in the presence of a DNA polymerase to form double-stranded DNA; (g) initiating transcription of the double-stranded DNA by denaturing the double-stranded DNA of step (f) in the presence of an RNA polymerase and dNTPs to form RNA amplification products; and (h) initiating reverse transcription of the RNA amplification products by reacting the RNA amplification products of step (g) with a reverse transcriptase in the presence of dNTPs to generate single stranded DNA.

The DNA used in the sample is usually, although not necessarily, genomic DNA, and the sample is usually, although not necessarily, a tissue sample. Tissue samples for use with the method may be microdissected tissue samples or formaldehyde-fixed tissue samples.

An agent for use in step (b) of the method is sodium bisulfite, although other agents that may convert methylated CpG sites may be used.

An enzyme for use in step (c) of the method is an alkaline phosphatase, although other agents that release phosphate groups may be used. One alkaline phosphatase that may be used in step (c) is calf intestinal alkaline phosphatase.

When the homopolymer tail of the modified DNA of step (d) is a poly(T) tail, the homopolymer tail of the primer of step (e) is a poly(A) tail.

Primers that may be used in step (e) include T7 promoter primer, SP6 promoter primer, and T3 promoter primer. An example of a polymerase that may be used in step (f) includes without limitation DNA polymerase 1 Klenow Fragment.

RNA polymerases that may be used in step (g) include T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase.

In another aspect of the invention, the amplification products obtained by the method are identified by an application selected from the group consisting of methylation specific primer extension, DNA microarrays, microbead assays, and a planar waveguide chips.

In a further aspect of the invention, the method is used to identify single nucleotide polymorphisms at CpG sites in the genome of an organism. Examples of genomes that may be screened using the method of the present invention may be selected from the group consisting of a haploid set of chromosomes in a eukaryotic species, a single chromosome in bacterial species, and DNA or RNA in a viral species.

Additional aspects, advantages and features of the invention will be set forth, in part, in the description that follows, and, in part, will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

BRICΓ DESCRIPTION OF THE DRAWINGS

Figure 1 is schematic diagram of the method of the present invention as used for the linear amplification of bisulfite converted DNA.

DETAILED DESCRIP TION OF I HE INVENTION

As used in this specification and the appended claims, the singular forms "a,^"' "'an,^"' and "the" include plural referents unless the context clearly dictates otherwise.

The definitions that follow are used for the purpose of describing particular embodiments of the invention and are not intended to be limiting.

The term "sample^*1 is meant to include any material containing DNA or RNA that is obtained from an animal, plant, bacterial, viral, or fungal species. Examples of samples that may be used in the method of the present invention include tissues or fluids obtained from a live source (i.e., an animal or human) or from in vitro cell culture. Tissue samples that may be used with the method of the present invention may be obtained, without limitation, from any of the following sources: skin; bone; muscles; tendons; cartilage; organs; respiratory, intestinal, or genitourinary tracts; and hair. Fluids that may be used with the method of the present invention include, without limitation, any of the following: blood, plasma, serum, cerebrospinal fluid, synovial fluid, lymph, tears, saliva, amniotic fluid, amniotic cord blood, mucus, urine, vaginal secretions, and semen.

The term "target" refers to a molecule, gene, or genome containing a nucleic acid sequence or sequence segment that is intended to be characterized by way of identification, quantification, or amplification. The term "gene" refers to a particular nucleic acid sequence within a DNA molecule that occupies a precise locus on a chromosome and is capable of self-replication by coding for a specific polypeptide chain. The term "genome'" refers to a complete set of genes in the chromosomes of each cell of a specific organism. Within the context of the present invention, the term "target gene^"' is used to refer to a gene to be analyzed in a sample and the term "genomic DNA'" refers to the full complement of DNA contained in the genome of a cell or a sample.

The term "methylation" generally refers to an enzyme-mediated chemical modification that adds methyl (CH₁) groups at selected sites on proteins, DNA, and RNA. In the context of the present invention, the term methylation refers to the methylation of the fifth carbon position of cytosines on CpG sites. As previously noted, in humans and most mammals, DNA methylation is the only known natural modification of DNA and is specific to cytosines at CpG sites.

The term "CpG" refers to a cytosine-guanine diniicleotide where the "p" stands for the phosphodiester bond between the two nucleotides. As noted previously, "CpG sites" are sites that typically have methylated cytosines and "CpG islands" are sites that do not typically have methylated cytosines. Methylation of cytosines on CpG islands has been found to be responsible for genomic imprinting disorders and the silencing of tumor suppressor genes, the former resulting in the expression of sex-linked disorders and the latter resulting in the formation of cancerous tumors.

The term "SNP" refers to a variant DNA sequence in which a purine or pyriinidine base of a single nucleotide has been replaced with another base. An example of an SNP is a DNA sequence where a cytosine has been replaced with a thymine in one single location of the sequence. SNPs are also referred to as single point mutations.

The term "terminal transferase" ("TdT") refers to a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3'-hydroxyl terminus of DNA molecules. Terminal transferases are used to add polynucleotide tails to the 3' ends of DNA. One type of polynucleotide tail that may be added to DNA is a homopolymer tail, which is constructed by reacting DNA with single nucleotide dNTPs in the presence of terminal transferase. The term "single nucleotide dNTPs^'' refers to a population of dNTPs, wherein N = a single nucleotide, such as for example, a population of dTTPs, or dATPs, etc.

The term "Klenow Fragment" refers to the large fragment of DNA polymerase I that exhibits 5'-3' polymerase activity and 3'-5' exonuclease activity and thus is able to displace downstream oligonucleotides as it polymerizes. When DNA polymerase I (which is isolated from Escherichia coll) is exposed to the protease subtilisin, it is cleaved into two fragments, a small fragment with 5'-3' exonuclease activity, and the large Klenow fragment with 3'-5' exonuclease activity.

The term "DNA polymerase" refers to an enzyme that catalyzes the synthesis of DNA in the 5'-3' direction. DNA polymerases require a primer to provide a free 3' hydroxyl group to initiate complementary strand synthesis from free dNTPs. The term ''RNA polymerase" refers to an enzyme that catalyzes the synthesis of RNA in the 5'-3' direction in the presence of a DNA template containing a promoter sequence.

The term "reverse transcription^" refers to the biochemical process to generate single-stranded DNA from a single-stranded RNA template using the DNA polymerase reverse transcriptase as a catalyst.

The term "linear amplification" refers to an amplification process that uses multiple cycles of primer extension reactions to amplify a target DNA. With linear amplification, the relative representation of each transcript species from the original sample is maintained both during and after amplification. An example of a linear amplification procedure is LCR, the aRNA method of Phillips and Eberwine, supra, and the linear amplification method described herein.

The term "exponential amplification^'' refers to an amplification procedure where the product (i.e., amplicon) doubles with every reaction cycle. An example of an exponential amplification procedure is PCR.

The term "primer" refers to an oligonucleotide, whether produced naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced, i.e., in the presence of appropriate nucleotides and an agent for polymerization such as a DNA polymerase in an appropriate buffer and at a suitable temperature.

The term "probe'^" refers to an oligonucleotide that forms a hybrid structure with a target sequence contained in a molecule (i.e., a "target molecule") in a sample undergoing analysis, due to complementarity of at least one sequence in the probe with the target sequence. The nucleotides of any particular probe may be deoxyribonucleotides, ribonucleotides, and/or synthetic nucleotide analogs.

The term "oligonucleotide" encompasses polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones (e.g., protein nucleic acids and synthetic sequence-specific nucleic acid polymers commercially available from the Anti-Gene Development Group. Corvallis, Oregon, as NEUGENE® polymers) or nonstandard linkages, providing that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, such as is found in DNA and RNA. In this respect, the term "oligonucleotide" includes double- and single-stranded DNA, double- and single-stranded RNA, DNA:RNA hybrids, and other types of modified oligonucleotides. Modified oligonucleotides include for example, oligonucleotides wherein one or more of the naturally occurring nucleotides is substituted with an analog; oligonucleotides containing internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), or positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters); oligonucleotides containing pendant moieties such as proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); oligonucleotides with intercalators (e.g., acridine, psoralen, etc.); oligonucleotides containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.); and oligonucleotides containing atkylators. There is no intended distinction in length between the terms "polynucleotide^"' and "oligonucleotide,^'' and these terms are used interchangeably to refer to the primary structure of the molecule. As used herein the symbols for nucleotides and polynucleotides are according to the IUPAC-I UBMB Joint Commission on Biochemical Nomenclature (see, http://www.chem.qmul.ac.uk/iupac/jcbn).

As used herein, the term '"homopolymer^" refers to a polymer comprised of a string of a single nucleotide, such as string of thymines or a string of adenines that is attached to the 3' end of a DNA or RNA molecule. Examples of homopolymer are the poly(T) and poiy(A) tails that are used in the linear amplification method of the present invention.

Oligonucleotides for use in the present invention may be synthesized by known methods. Background references that relate generally to methods for synthesizing oligonucleotides include those related to 5'-to-3' syntheses based on the use of β-cyanoethyl phosphate protecting groups. See, e.g , de Napoli et al., GAZZ CH(M I I ΛL 1 14:65 ( 1984); Rosenthal et al., Tt 1 RΛHLDRON LETT 24: 1691 (1983); Belagaje and Brush, NUC ACIDS RCS 10:6295 ( 1977); in references which describe solution- phase 5'-to-3' syntheses include Hayatsu and Khorana, J AM CΠEM SOC 89:3880 (1957); Gait and Sheppard, Nυc ACIDS RES 4: 1 135 ( 1977); Cramer and Koster, ANGEW CHEM INT ED ENGL 7:473 (1968); and Blackburn et al., J CHEM SOC PART C, at 2438 ( 1967). Additionally, Matteucci and Caruthers, J AM Cl ICM Soc 103:3185-91 ( 1981) describes the use of phosphochloridites in the preparation of oligonucleotides; Beaucage and Caruthers, TETRAHEDRON LETT 22: 1859-62 (1981), and U.S. Pat. No. 4,415,732 to Caruthers et al. describes the use of phosphoramidites for the preparation of oligonucleotides. Smith, AM BlOTRCH LAB, pp. 15-24 (December 1983) describes automated solid-phase oligodeoxyribonucleotide synthesis; and T. Horn and M.S. Urdea, DNA 5:421- 25 ( 1986) describe phosphorylation of solid-supported DNA fragments using bis(cyanoethoxy)-N,N- diisopropylaminophosphine. See also, references cited in Smith, supra; Warner et al., DNA 3:401- 1 1 (1984); and T. Horn and M.S. Urdea, TE I RAl IEDRON LEΠ . 27:4705-08 ( 1986).

The term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. Expressed Sequence Tags ("ESTs," i.e., small pieces of DNA sequence usually 200 to 500 nucleotides long generated by sequencing either one or both ends of an expressed gene), chromosomes, cDNAs, niRNAs, and rRNAs are representative examples of molecules that may be re i erred to as nucleic acids.

The terms "nucleotide" and "nucleoside" refer to nucleosides and nucleotides containing not only the four natural DNA nucleotidic bases, i.e., the purine bases guanine (G) and adenine (A) and the pyrimidine bases cytosine (C) and thymine (T), but also the RNA purine base uracil (U), the non- natural nucleotide bases iso-G and iso-C, universal bases, degenerate bases, and other modified nucleotides and nucleosides. Universal bases are bases that exhibit the ability to replace any of the four normal bases without significantly affecting either melting behavior of the duplexes or the functional biochemical utility of the oligonucleotide. Examples of universal bases include 3- nitropyrrole and A-, 5-, and 6-nitroindole, and 2-deoxyinosine (dl), that latter considered the only "natural" universal base. While dl can theoretically bind to all of the natural bases, it codes primarily as G. Degenerate bases consist of the pyrimidine derivative 6H.8H-3,4-dihydroρyrimido[4,5- c][l ,2]oxazin-7-one (P), which when introduced into oligonucleotides base pairs with either G or A, and the purine derivative N6-methoxy-2,6,-diaininopurine (K), which when introduced into oligonucleotides base pairs with either C or T. Examples of the P and K base pairs include P-imino, P-amino, K-imino, and K-amino.

The terms '"complementary" and "substantially complementary" refer to base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), and G and C. Within the context of the present invention, it is to be understood that the specific sequence lengths listed are illustrative and not limiting and that sequences covering the same map positions, but having slightly fewer or greater numbers of bases are deemed to be equivalents of the sequences and fall within the scope of the invention, provided they will hybridize to the same positions on the target as the listed sequences. Because it is understood that nucleic acids do not require complete complementarity in order to hybridize, the probe and primer sequences disclosed herein may be modified to some extent without toss of utility as specific primers and probes. Generally, sequences having homology of 80% or more fall within the scope of the present invention. As is known in the art, hybridization of complementary and partially complementary nucleic acid sequences may be obtained by adjustment of the hybridization conditions to increase or decrease stringency, i.e., by adjustment of hybridization temperature or salt content of the buffer. Such minor modifications of the disclosed sequences and any necessary adjustments of hybridization conditions to maintain specificity require only routine experimentation and are within the ordinary skill in the art.

The term "hybridizing conditions" is intended to mean those conditions of time, temperature, and pH, and the necessary amounts and concentrations of reactants and reagents, sufficient to allow at least a portion of complementary sequences to anneal with each other. As is well known in the art, the time, temperature, and pH conditions required to accomplish hybridization depend on the size of the oligonucleotide probe or primer to be hybridized, the degree of complementarity between the oligonucleotide probe or primer and the target, and the presence of other materials in the hybridization reaction admixture. The actual conditions necessary for each hybridization step are well known in the art or can be determined without undue experimentation. Typical hybridizing conditions include the use of solutions buffered to a pH from about 7 to about 8.5 and temperatures of from about 3O⁰C to about 60⁰C, preferably from about 37⁰C to about 55°C for a time period of from about one second to about one day, preferably from about 15 minutes to about 16 hours, and most preferably from about 15 minutes to about three hours. Hybridization conditions also include a buffer that is compatible, i.e., chemically inert, with respect to primers, probes, and other components, yet still allows for hybridization between complementary base pairs, can be used. The selection of such buffers is within the knowledge of one of ordinary skill in the art.

It is understood by one of ordinary skill in the art that the isolation of DNA and RNA target sequences from a sample requires different hybridization conditions. For example, if the sample is initially disrupted in an alkaline buffer, double stranded DNA is denatured and RNA is destroyed. By contrast, if the sample is harvested in a neutral buffer with SDS and proteinase K, DNA remains double stranded and cannot hybridize with the primers and/or probes and the RNA is protected from degradation.

The terms "'support" and "substrate" are used interchangeably to refer to any solid or semisolid surface to which an oligonucleotide probe or primer, analyte molecule, or other chemical entity may be anchored. Suitable support materials include, but are not limited to, supports that are typically used for solid phase chemical synthesis such as polymeric materials and plastics for use in beads, sheets, and microtiter wells or plates examples including without limitation, polystyrene, polystyrene latex, polyvinyl chloride, polyvinylidene fluoride, polyvinyl acetate, polyvinyl pyrrolidone, polyacrylonitrile, polyacrylamide, polymethyl methacrylate, polytetrafluoroethylene, polyethylene, polypropylene, polycarbonate, and divinylbenzene styrene-based polymers; polymer gels; agaroses such as SEPHAROSE®; dextrans such as SEPHADEX®); celluloses such as nitrocellulose; cellulosic polymers; polysaccharides; silica and silica-based materials; glass (particularly controlled pore glass) and functional ized glasses; ceramics, and metals. Preferred supports are solid substrates in the form of beads or particles, including microspheres, nanospheres, microparticles, and nanoparticles.

The term '"label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, and that can be attached to a nucleic acid or protein via a covalent bond or noncovalent interaction (e.g., through ionic or hydrogen bonding, or via immobilization, adsorption, or the like). Labels generally provide signals detectable by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. Examples of labels include fluorophores, chromophores, radioactive atoms (particularly ¹²P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. Within the context of the present invention, fluorophores will be the most commonly used label to identify, quantify, and analyze the linearly amplified DNA products of the present invention. Examples of fluorophores include without limitation, Examples of fluorophores that may be used with PWG chips include without limitation, fluorescein dyes (e.g., fluorescein isothiocyanate ("FITC")), rhodamine dyes, eosin dyes, cyanine dyes (e.g., allophycocyanin), SYBR® green dye (Molecular Probes, Eugene, OR), BOD1PY® dye (Molecular Probes, Eugene, OR), TEXAS RED® dye (Molecular Probes, Eugene, OR), CY CHROME™ dye, phycoerythrin, and streptavidin, the latter two of which are frequently used in a complex (see, Example 3).

The term "singleplex" refers to a single assay that is not carried out simultaneously with any other assays. Singleplex assays include individual assays that are carried out sequentially.

The term '"multiplex^" refers to multiple assays that are carried out simultaneously, in which detection and analysis steps are generally performed in parallel. Multiplex assays are typically hybridization assays.

Set forth below is a description of what are currently believed to be the preferred embodiments and best examples of the claimed invention. Any alternates or modifications in function, purpose, or structure are intended to be covered by the claims of this application.

The present invention describes a method for preserving, linearly amplifying, and labeling bisulfite converted DNA that overcomes many of the shortcomings inherent in the amplification procedures presently used in the art to amplify bisulfite converted DNA. Specifically, the L-ABCD method of the present invention eliminates the PCR bias inherent in bisulfite DNA PCR; does not require the use of target specific primers, which are required with HAD; and does not require high molecular DNA as does MDA. The L-ABCD method has the advantages of being able to amplify DNA from very small samples and/or from samples that contain degraded DNA.

In one embodiment of the invention, there is provided a method of linearly amplifying bisulfite converted DNA comprising the steps of: (a) obtaining DNA from a sample to be analyzed and denaturing the DNA to form single-stranded DNA; (b) generating modified DNA by reacting the single-stranded DNA with bisulfite, wherein the bisulfite converts unmethylated cytosine residues in the DNA to uracil while leaving methylated cytosine residues unchanged; (c) treating the modified DNA with an enzyme that releases phosphate groups from 3' ends of the modified DNA; (d) adding a homopolymer tail to the 3' end of the modified DNA by reacting the modified DNA with single nucleotide dNTPs in the presence of an enzyme; (e) annealing a primer to the homopolymer tail of the modified DNA, wherein the primer has a homopolymer tail that is complementary to the homopolymer tail of the modified DNA; (0 initiating DNA replication by reacting the modified DNA of step (e) with dNTPs in the presence of a DNA polymerase to form double-stranded DNA; and (g) initiating transcription of the double-stranded DNA by denaturing the double-stranded DNA in the presence of an RNA polymerase and dNTPs to form RNA amplification products; and (h) initiating reverse transcription of the RNA amplification products by reacting the RNA amplification products with a reverse transcriptase in the presence of dNTPs to generate single stranded DNA.

If additional amplification is desired, steps (c) to (e) are repeated to generate the desired number of linearly amplified RNA species, which are subsequently transcribed to single-stranded DNA or double-stranded DNA when further reacted with DNA polymerase in the presence of a suitable primer and dNTPs.

The DNA for use with the linear amplification method of the present invention will typically be genomic DNA; however, non-genomic DNA, such as mitochondrial DNA, ESTs, and viral DNA may also be used with the method of the present invention.

Currently, the only known agent used to modify the DNA in step (b) is sodium bisulfite; however, the invention contemplates that other agents may be used to modify the DNA as they become known.

An enzyme that may be used to release the phosphate groups from the 3' ends of the single- stranded DNA in step (c) is calf intestinal alkaline polypeptide ("ClP^*7); however, any alkaline phosphatase or other enzyme that prevents the self-ligation of single-stranded DNA may be used for this step.

Examples of an enzyme and homopolymer combination that may be used for step (d) include terminal transferase ("TdT") and dTTP, respectively; however, it is to be understood that other homopolymer tails may be added to the 3' end of the single-stranded DNA of step (d). The length of the homopolymer tail may range from approximately 10 nucleotides to 50 nucleotides; a 20- nucleotide tail is frequently used for most molecular biology applications.

Any suitable primer and may be used for step (e). Examples of suitable primers that may be used in step (e) are any of the following primers:

T7 promoter primer (20-mer):

5'-d(TAA TAC GAC TCA CTA TAG GG)-3' (SEQ ID NO. 1 )

SP6 promoter primer (24-mer):

5^r-d(CAT ACG ATT TAG GTG ACA CTA TAG)-3' (SEQ ID NO.2)

T3 promoter primer (23-mer):

5'-d(GCA ATT AAC CCT CAC TAA AGG GA)-3' (SEQ ID NO.3)

As is noted above, the primer of step (e) will have a homopolymer tail attached at the 3' end of the primer, which is complementary to the homopolymer tail attached at the 3' end of the modified DNA. To facilitate the DNA replication reaction of step (f), the homopolymer tail of the primer may be anchored with a 3' base that differs from the base of the homopolymer tail (i.e., a degenerate base); for example, where the homopolymer tail on the modified DNA is a poly(T) tail and the homopolymer tail on the primer is a po!y(A) tail, the homopolymer tail of the primer may be anchored with a nucleotide that is C, G, or T.

The DNA replication of step (f) may be initiated by any suitable DNA polymerase, such as for example, the Klenow fragment of DNA polymerase 1.

The RNA polymerase that will be used for the transcription of step (g) will be dependent on the choice of primer selected, thus for example, where the T7 promoter primer is used, T7 polymerase is the RNA polymerase; where the SP6 promoter primer is use, SP6 polymerase is the RNA polymerase; and where the T3 promoter primer is used, T3 polymerase is the RNA polymerase.

Any known reverse transcriptase may be used for step (g). One commonly used reverse transcriptase is the IMPROM-II™ reverse transcriptase commercially available from Promega Biosciences, Inc., San Luis Obispo, CA.

As indicated above, the homopolymer tail of the primer may be anchored with a degenerate base.

The L-ABCD method has utility in the interrogation of the methylation status of DNA at CpG sites and at SNP sites. The use of the assay to interrogate CpG islands is particularly useful in that it enables the analysis of the epigenomic status of genes that may differ between normal and disease states. The use of the assay to interrogate SNP sites is also particularly useful in that it allows for the identification of single point mutations that are the result of DNA methylation.

Conventional assays known in the art for interrogating CpG sites use bisulfite to convert the unmethylated cytosines in the DNA to uracil; however, the bisulfite has the deleterious consequence of degrading the majority of the DNA in the sample. Because the conventional bisulfite assays are not capable of amplify ing the degraded DNA, it follows that the conventional bisulfite assays only have utility on the remaining non-degraded DNA in a bisulfite-treated sample, which is generally an amount sufficient for not more than a few assays. In light of the foregoing, it follows that the conventional bisulfite assays are not suitable for interrogating multiple CpG target sites. Further, because the conventional bisulfite assays are incapable of interrogating CpG sites from samples that do not have large quantities of high quality DNA, it follows that the conventional bisulfite assays also have little to no utility for interrogating CpG sites from very small samples and/or from degraded samples.

Unlike the conventional bisulfite assays, the L-ABCD method of the present invention propagates the bisulfite converted DNA before proceeding into the assay. In this way, the L-ABCD method of the present invention is capable of preserving the DNA from small and/or degraded samples. Because the L-ABCD method is able to preserve DNA from small and degraded DNA samples, the L-ABCD method is capable of interrogating CpG sites in degraded DNA from very small samples. The L-ABCD method of the present invention also allows for the subsequent amplification and/or labeling of the DNA for downstream applications.

Examples of small and/or degraded samples that have utility with the L-ABCD method of the present invention include without limitation microdissected tissue, fine needle aspirate, and FFPE tissue (i.e., formalin-fixed paraffin-embedded tissue), the latter being a highly crosslinked and degraded tissue due to the exposure of the tissue samples to formaldehyde during the fixation process. Generally, nucleic acids that are formaldehyde fixed will have DNA fragments in the range of 300- 400 bp and RNA fragments in the range of 200 bp (see e.g., Lehmann and Kreipe, METHODS 25:409- 418, 410 (2001 ).

Because the L-ABCD method of the present invention is capable of amplifying highly degraded single-stranded DNA, it follows that the method does not require high quality high molecular weight DNA. In this respect, the L-ABCD method of the present invention is particularly advantageous because it is capable of interrogating DNA universally in a whole genome approach. This aspect of the present invention is particularly important in the genomic screening of genetic variations, such as SNPs, where a low density of SNPs in a genome (e.g. 2% in a genome) may not indicate a disease state whereas a higher density of SNPs in a genome (e.g., 25% or greater) may indicate a disease state or a high risk of disease. In this respect, if an SNP screen is limited to a candidate region of a sample where all of the SNPs are populated, a patient's disease susceptibility may be calculated to be significantly higher than it actually may be. The L-ABCD method of the present invention overcomes this shortcoming in the art by allowing a whole genome approach to interrogating SNPs or other genetic variations. Examples of genomes that may be screened for SNPs using the L-ABCD method of the present invention include a haploid set of chromosomes in a eukaryotic species, a single chromosome in bacterial species, and DNA or RNA in a viral species.

Downstream applications that may be used to assay the amplification products of the L- ABCD method of the present invention include without limitation, PCR (Example 3), the methylation specific primer extension ("MSPE") assay described in co-owned, co-pending U.S. Patent Publication No. 2005/0214812 to Li and Harvey, incorporated by reference in its entirety herein (Example 4 describes the MSPE assay used with a PCR platform, i.e., MS-PCR); DNA microarray assays; microbead assays, such as the LUMINEX® microbead platform (Luminex Corp., Austin, TX) (Example 5); and planar waveguide ('"PWG") chips, which are described in co-owned, co-pending U.S. Patent Application entitled "Planar Waveguide Detection Chips and Chambers for Performing Multiplex PCR Assays" to Warner et al. filed on November 14, 2005, incorporated by reference in its entirety herein.

The MSPE assay of Li and Harvey identifies amplified methylated DNA using at least one methylation specific primer that hybridizes to the amplified methylated DNA in the presence of labeled dNTPs and a DNA polymerase; thus, following the linear amplification method of the present invention, the MSPE assay may be used to identify the methylation status of the amplification products,

In one embodiment of the single primer MSPE assay, the most 3'end of the primer hybridizes to the cytosine residue of the CpG site to be analyzed on the top strand of the methylated DNA while the 5' end of the primer has a unique sequence that does not hybridize to any sequences in the DNIA sample. In another embodiment of the MSPE assay, one MSPE primer is used that has a polynucleotide sequence at the most 3¹ end of the primer that hybridizes to the guanine residue complementary to the cytosine residue on the CpG site to be analyzed on the bottom strand of the methylated DNA while the 5' end of the primer has a unique sequence that does not hybridize to any sequences in the DNA sample.

Where two MSPE primers are used, one of the primers is specific for methylated DNA while the other is specific for unmethylated DNA. In one embodiment of the dual primer MSPE assay, where the most 3' end of the first primer hybridizes to the cytosine residue of the CpG site to be analyzed while the 5' end has a unique first sequence that does not hybridize to any sequences in the sample, the most 3' end of the second primer hybridizes to the thymine residue on the top stand of the unmethylated DNA, which is derived from the cytosine of the CpG site to be analyzed, while the 5' end of the second MSPE primer has a unique second sequence that does not hybridize to any sequences in the DNA sample. In another embodiment, where the most 3' end of the first primer hybridizes to the guanine residue complementary to the cytosine residue on the CpG site to be analyzed while the 5' end has a unique first sequence that does not hybridize to any sequences in the sample, the most 3' end of the second primer hybridizes to the adenine residue on the bottom strand of the unmethylated DNA, which is derived from the cytosine of the CpG site on the top strand, while the 5' end of the second MSPE primer has a unique second sequence that does not hybridize to any sequences in the DNA sample.

After the primer extension products described above are hybridized to at least one pair of oligonucleotides wherein the first oligonucleotides is complementary to the first unique sequence and the second oligonucleotide in the pair is complementary to the second unique sequence, the 5' methylation status at the cytosine residue of the CpG site is determined by comparing the hybridization intensity of the methylated DNA to the hybridization intensity of the unmethylated DNA in the sample.

DNA microarrays provide a medium for matching known and unknown DNA samples through base pairing and automated identification of unknowns. Generally, array protocols involve manual or robotic depositing of oligonucleotide probes of known sequence on a substrate, which is usually made of glass or nylon. The sequences of unknown DNA samples are identified upon hybridization of the target nucleic acid sequences to the known probe sequences. See, Ekins and Chu, TRCNDS [N BlO I LCHNOLOGY 17:217-218 ( 1999). In general, arrays are described as "macroarrays" or '"microarrays,'^" the difference being the size of the sample spots. While macroarrays contain sample spot sizes of about 300 microns or larger that can be imaged by gel and blot scanners, the sample spot sizes in microarrays are typically less than 200 microns in diameter. Because microarrays usually contain thousands of spot, specialized robotics and imaging equipment are required to generate the arrays. Within the context of the present invention, the linearly amplified DNA product may be identified by hybridizing the amplified DNA to probes of known sequences on a suitable substrate such as a multiwell plate or beads.

The LUM1NEX® microbead platform captures amplified DNA products on color-coded polystyrene microbeads that are internally dyed with red and infrared fluorophores. By using different intensities of the two dyes for different batches of microspheres, up to 100 microsphere sets may be produced, each with a unique spectral signature, which is determined by the red/infrared signature of the microsphere sets; the unique spectral signature of the bead sets allows for the multiplexing of up to 100 different samples. By coupling the LUMINEX® bead with probes that are complementary to a target sequence of interest and that are also tagged with a reporter dye that is excitable by green light, the beads may be used to identify target sequences of interest in a multiplex format by the subsequent analysis of the red/infrared dyes in the beads and the reporter dyes coupled to the beads. The most common reporter dye used with the LUMINEX® platform is phycoerythrin or streptavidin-phycoerythrin (see, Example 3). The identification process is carried out with a flow cytometer that uses a red laser to excite the red/infrared dyes and thereby classify the spheres (i.e., the classification channel reading) and a green laser that excites any orange fluorescence associated with the reporter dye (i.e., the reporter channel reading); under this system, each bead is subject to a classification and reporter channel reading. Within the context of the present invention, the linearly amplified DNA may be identified and quantified by binding a target sequence of interest of the amplicon to an oligonucleotide that is bound to a LUMINEX® microsphere that is tagged with a reporter dye.

PWG technology combines highly selective fluorescence detection with high sensitivity. PWGs are 150 to 300 nm thin films made of a material with a high refractive index, such as titanium dioxide (TiO₂) or tantalum pentoxide (Ta₂O₅), that are deposited on a transparent support with a low refractive index, such as glass, silicon dioxide, or a polymer. A parallel laser light beam is coupled into the waveguiding film by a diffractive grating that is etched or embossed into the substrate. When the light propagates within the film, a strong evanescent field that is perpendicular to the direction of propagation is produced, which enters into the adjacent medium. The intensity of he evanescent filed can be enhanced by increasing the refractive index of the waveguiding layer and decreasing the layer thickness. Compared to confocal excitation of the field intensity close to the surface can be increased by a factor or up to 100. The field strength decays exponentially with the distance from the waveguide surface and its penetration depth is limited to about 400 nm. This effect can be used to selective excite only fluorophores located at or near the surface of the waveguide, which in turn results in a significant decrease in background interference that results from fluorescence emission of the solution in the well.

When PWG technology is used for bioanalytical applications, specific capture probes or recognition elements for the analyte of interest are immobilized on the PWG surface. The presence of the analyte in a sample applied to a PWG chip is detected using fluorophores (i.e., fluorescent dyes) attached to the analyte or one of its binding partners in the assay. Upon fluorescence excitation of the evanescent field, excitation, and detection of fluorophores is restricted to the sensing surface, while signals from unbound molecules in the bulk solution are not detected. The result is an increase in the signal to noise ratio in microarrays over conventional optical detection methods. Within the context of the present invention, the linearly amplified DNA products may be analyzed by labeling the DNA sample with a fluorescent dye and contacting the DNA sample with oligonucleotide probes captured on the PWG surface.

It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

All patents and publications mentioned herein are incorporated by reference in their entireties.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions of the invention. The examples are intended as non-limiting examples of the invention. While efforts have been made to ensure accuracy with respect to variables such as amounts, temperature, etc., experimental error and deviations should be taken into account. Unless indicated otherwise, parts are parts by weight, temperature is degrees centigrade, and pressure is at or near atmospheric. All components were obtained commercially unless otherwise indicated.

EXPERIMENTAL

The practice of the present invention will use, unless otherwise indicated, conventional techniques of molecular biology, biochemistry, microbiology, and the like, which are within the skill of the art. In the examples that follow, efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but experimental error and deviations should be taken into account when conducting the described experiments. Unless indicated otherwise, parts are parts by weight, temperature is degrees centigrade, and pressure is at or near atmospheric. All components were obtained commercially unless otherwise indicated.

The following protocol is used to isolate DNA from FFPE tissue samples: One to six 10 μm sections are cut from a paraffin block and placed in a 1 .5 mL screw cap tube without deparaffinization or rehydration along with 850 μL denaturation solution (4 M guanidinium isothyiocyanate, 0.25 M sodium citrate, 0.5% sarcosyl. and 0.1 M 2-mercaptoethanol) and 250 μL proteinase K (20 mg/mL in water). The samples are incubated overnight at 55⁰C with vigorous agitation (e.g., 1400 rpm). After the incubation period is over, the tubes are centrifuged at 4°C for 5 minutes at 14,000 g at which time a white cap of solidified paraffin will form on the surface of the solution. Next, the digested samples are transferred to a clean 2 mL safelock tube; at this stage, it is important not to transfer any undigested material and/or solidified paraffin. The following solution is added to the sample tube, with careful mixing: 100 μL 3 M sodium acetate (pH 5.2), 630 μL water- saturated phenol, and 270 μL chloroform. The sample tube is placed on ice for 15 minutes and then centrifuged at 4°C for 20 minutes at 14,000 g. Approximately 1 mL of the upper aqueous phase is removed to a 2 mL tube, while avoiding the transfer of any material collected at the interface, and the following mixture is added to the sample: 1 μL glycogen and 1 mL isopropanol. The samples are then stored overnight at -20°C, centrifuged at 4^QC for 20 minutes at 14,000 g, the pellet is washed once with 70% ethanol, and allowed to air dry. The air-dried pellet is then dissolved in 20 to 100 μL diethyl pyrocarbonate treated water, being careful to not over dry the pellet, and used directly or stored for several weeks at -20⁰C.

The following protocol is used to isolate DNA from microdissected samples: The tissue sample is placed into a 0.5 mL reaction tube with 3O-5OμL of the following lysis buffer: 50 mM Tris-HCi (pH 8.1), 1 mM EDTA, 0.5% Tween 20, and 0.1 mg/mL proteinase K. The samples are inverted and incubated overnight at 40°C. After the incubation period is over, the tubes are centrifuged for 3 minutes at 14,000 g, incubated at 95°C for 10 minutes to inactivate the proteinase K, and centrifuged briefly and used directly or stored at -20⁰C.

EXΛMI'LH I

BISULFITE CONVΓ.RSION OF GENOMIC DNA

Genomic DNA is extracted from a microdissected sample or an FFPE tissue sample using the protocols described above, respectively, and then treated with sodium bisulfite to convert all unmethylated cytosines to uracil while leaving all 5' unmethylated cytosines intact according to the procedure set forth in Frommer et al., PROC NAΪ 'L ACΛD SCI USA 89: 1827- 1831 (1992). The procedure for producing purified bisulfite converted DNA is as follows:

1 μg of genomic DNA is placed in a 1 mL reaction tube and diluted with distilled water and 5.5μL of 2M NaOH and incubated for 10 minutes at 37°C to generate single stranded DNA;

30 μL of freshly prepared 10 mM hydroquinone is added to the tube of DNA followed by 520μL of freshly prepared 3M sodium bisulfite, pH 5.0; and reagents ai e thoroughly mixed, covered with mineral oit and incubated foi 50⁰C for 16 hours at which time the DNA is conveited (at longer duration, methylated cytosine starts to conveit to thymine).

At the end of the incubation period, the oil is l emoved and the DNA solution is diluted with an appropriate solution for column purification. After column purification, the DlNlA is precipitated, washed with 70% ethanol, and dried and suspended in 20 μL water.

EXΛMPLt 2

LINLΛR AMPI IΠCA FiON OF Bisui ΠTΓ CONVL R I LD DNA

The single-stianded bisulfite converted DNA from Example 1 is reacted at 37°C with TdT (terminal transfeiase) in the presence of dTTP (2'-deoxythymidine 5'-triρhosρhate) to generate a 20-40 bp polyT tail on the 3' end of the DNA strand

A T7-(A)₁₈B anchored primer adaptor, where B = C, G, or T, is annealed to the 3' polyT tail of the DNA strand.

A second strand of DNA is geneiated using DNA polymerase I Klenow Fragment and dNTPs. The resulting double-stranded DNA is identical to the original genomic DNA except that the original strand has a T7 promoter at the end of the 3' poly(T) tail and the second sti and has a T7 promoter at the end of the 5' po!y(A) strand.

The double-stranded DNA is then denatured by heating and in vitro transcription is initiated by reacting the denatured DNA with T7 RNA polymerase at 37°C.

The resulting RNA amplification product is then ieacted with a reverse transcriptase enzyme to form single stranded DNA, which may be further replicated to form double-stranded DNA.

EXΛMPLL 3 PCR AMPLM (CATION FROM DNA GrNCRΛ I LD \ ROM 11 [L L-ABCD MLl I IOD

To obtain a large quantity of DNA from the product of Example 2, the double-stranded DNA may be subjected to PCR

One microliter ( 1 μL) of DNA generated from the L-ABCD method of Example 2 (either single-stranded or double-stranded) is subjected to PCR amplification according to the following protocol.

The following pi imers are used for the PCR amplification.

Forward (p l όHmodl F): 5' GAA GAA AGA GGA GGG GTT GG 3' (SEQ lD NO. 4); and

Reverse (p l όHmod I R)' 5' CTA CAA ACC CTC TAC CCA CC 3' (SEQ ID NO. 5)

The PCR assay is conducted with a 50 μL sample containing the following ingredients: 20 iig of L-ABCD generated DNA; 15 mM Tris-HCl (pH 8.0); 2.0 mM MgCl₂; 50 mM KCl; 200 μM of each dNTP (i.e.. dATP, dCTP, dGTP, and dTTP); 0.4 μM final concentration of the forward PCR primer; 0.4 μM final concentration of the reverse PCR primer; and I unit of DNA Polymerase (e.g., AmpliTaq Gold® DNA Polymerase, Roche Molecular Systems, Inc., Alameda, CA)).

The following PCR cycle is used to conduct the PCR amplification: initial denaturation of 95°C for 10 minutes followed by a cycle of 95⁰C for 30 seconds; 6O⁰C for 30 seconds; and 72°C for 30 seconds, for a total of 40 cycles.

Amplicons generated from the PCR assay are analyzed by gel electrophoresis or quantified using a quantifying instrument, such as the Agilent Bioanalyzer (Palo Alto, CA).

EXAMPLE 4 ME I ΠYLA ΠON-SPL-CII-ΪC (MS) PCR ANALYSIS FOR UNA GENERATED FROM L-ABCD ML ITIOD

The reaction product of Example 2 may also be subjected to methylation specific primer extension in order to determine the methylation status of the amplification products.

One microliter ( 1 μL) of DNA generated from the L-ABCD method of Example 2 (either single-stranded or double-stranded) is subjected to methylation specific PCR ("MS-PCR") amplification according to the following protocol:

The following methylation specific primers are used for the MS-PCR amplification.

Forward (pi 6HBSM3) 5' GAG GGT GGG GCG GAT CGC 3' (SEQ lD NO. 6); and

Reverse (pl 6HBSM2) 5' GAC CCC GAA CCG CGA CCG TAA 3' (SEQ ID NO. 7).

The following unmethylation-specific primers are used for the MS-PCR amplification.

Forward (pi 6UF) 5' TTA TTA GAG GGT GGG GTG GAT TGT 3' (SEQ lD NO. 8); and

Reverse (pi 6UR) 5' CAA CCC CAA ACC ACA ACC ATA A 3' (SEQ ID NO. 9).

The PCR assay is conducted with a 50 μL sample containing the following ingredients: 20 ng of L-ABCD generated DNA; 15 mM Tris-HCl (pH 8.0); 2.0 inM MgCl₂ 50 mM KCI; 200 μM of each of dNTP (i.e., dATP, dCTP, dGTP, and dTTP); 0.4 μM final concentration of each of the four primers identified above; and 1 unit of DNA Polymerase (e.g., AmpliTaq Gold® DNA Polymerase, Roche Molecular Systems, Inc., Alameda, CA).

The following PCR cycle is used to conduct the MS-PCR amplification: initial denaturation of 95°C for 10 minutes followed by a cycle of 95°C for 30 seconds, 60⁰C for 30 seconds, and 72⁰C for 30 seconds, for a total of 40 cycles.

Amplicons generated from the PCR assay are analyzed by gel electrophoresis or quantified using a quantifying instrument, such as the Agilent Bioanalyzer (Palo Alto, CA). EXΛMPΪX 5 ANALYSIS OF LlNCΛRLY AMPLIFIED BlSULFI I E CONVERTED DNA USING LUMINF.X® BEADS

After the DNA amplification is complete (see, Examples 2, 3, and 4) and the DNA is labeled with for example, biotin. the DNA is transferred to a bead pool containing LUMIN EX® beads conjugated with amine-derivatized oligonucleotides and the beads were resuspended at 5000 μL in I x TE buffer (10 mM Tris, 1 mM EDTA, pH 8.5). Hybridization of the beads to the DNA is conducted in a reaction tube with 50μL hybridization reaction containing 0.5x hybridization buffer and LUM1NEX® beads. After incubation at 96⁰C for 2 minutes and 40⁰C for 30 minutes, the beads are washed once with 100 and 200 μL washing buffer (0.4x SSC, 1.0 g/L SDS, 0.50 g/L sodium azide (NaN₁), 0.50 g/L PROCLlN-300®, pH 7.7 (Rohm and Haas, Philadelphia, PA)) followed by a 15 minute reaction with 50 μL of 1 :500 diluted streptavidin-phycoerythrin dye (SA-PE, 1 mg/mL) at room temperature with gentle mixing, a final wash with 100 μL washing buffer, resuspension of the beads in 80 μL TTL buffer (50 mM Tris, 0.1 % Tween-20, 400 mM LiCl, pH 8.0), and the final qualitative and quantitative analyses of the DNA on a LUMlNEX- ] 00® instrument (Luminex Corporation, Austin, TX).

Claims

1 CLAIM:

1. A method of linearly amplifying bisulfite converted DNA comprising the steps of:

(a) obtaining DNA from a sample to be analyzed and denaturing the DNA to form single- stranded DNA;

(b) generating modified DNA by reacting the single-stranded DNA with bisulfite, wherein the bisulfite converts unmethylated cytosine residues in the DNA to uracil while leaving methylated cytosine residues unchanged;

(c) treating the modified DNA with an enzyme that releases phosphate groups from 3' ends of the modified DNA;

(d) adding a homopolymer tail to the 3^f end of the modified DNA by reacting the modified DNA with single nucleotide dNTPs in the presence of an enzyme;

(e) annealing a primer to the homopolymer tail of the modified DNA, wherein the primer has a homopolymer tail that is complementary to the homopolymer tail of the modified DNA;

(f) initiating DNA replication by reacting the modified DNA of step (e) with dNTPs in the presence of a DNA polymerase to form double-stranded DNA;

(g) initiating transcription of the double-stranded DNA by denaturing the double-stranded DNA of step (f) in the presence of an RNA polymerase and dNTPs to form RNA amplification products; and

(h) initiating reverse transcription of the RNA amplification products by reacting the RNA amplification products of step (g) with a reverse transcriptase in the presence of dNTPs to generate single stranded DNA.

2. The method of claim 1 , wherein the DNA is genomic DNA.

3. The method of claim 1 , wherein the sample is a microdissected tissue sample.

4. The method of claim 1 , wherein the sample is a formaldehyde-fixed tissue sample.

5. The method of claim 1 , wherein the agent of step (b) is sodium bisulfite.

6. The method of claim 1 , wherein the enzyme of step (c) is an alkaline phosphatase.

7. The method of claim 6, wherein the alkaline phosphatase is calf intestinal alkaline phosphatase.

8. The method of claim 1 , wherein the homopolymer tail of the modified DNA of step (d) is a poly(T) tail.

9. The method of claim 1 , wherein the primer of step (e) is selected from the group consisting of T7 promoter primer, SP6 promoter primer, and T3 promoter primer.

10. The method of claim 9, wherein the homopolymer tail of the primer is a poly(A) tail.

1 1. The method of claim 1 , wherein the DNA polymerase of step (f) is DNA polymerase 1 Klenow Fragment.

12. The method of claim 1 , wherein the RNA polymerase of step (g) is selected from the group consisting of T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase.

13. The method of claim 1 , wherein the amplification products are identified by an application selected from the group consisting of a methylation specific primer extension, DNA microarrays, microbead assays, and planar waveguide chips.

14. The method of claim 1 , used to screen for single point mutations in the genome of an organism.

15. The method of claim 14, wherein the genome is selected from the group consisting of a haploid set of chromosomes in a eukaryotic species, a single chromosome in bacterial species, and DNA or RNA in a viral species.