WO2008143367A1

WO2008143367A1 - Haplotyping method by multiplex amplification

Info

Publication number: WO2008143367A1
Application number: PCT/KR2007/002458
Authority: WO
Inventors: Jong Yoon Chun; Dae Hoon Lee; Byung Ha Jeon; Jong Kee Kim
Original assignee: Seegene, Inc.
Priority date: 2007-05-21
Filing date: 2007-05-21
Publication date: 2008-11-27

Abstract

The present invention relates to a method for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites and a kit for conducting such method. The present invention enables to simultaneously identify at least two types of nucleotide variations in at least two separate sites in higher fidelity, contributing to haplotyping of a DNA molecule in a single multiplex amplification. Even though the present invention is carried out in a relatively simple fashion, the results for haplotyping of genes are shown to be much more accurate than any conventional methods.

Description

HAPLOTYPING METHOD BY MULTIPLEX AMPLIFICATION

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for determining the haplotype of a

DNA molecule comprising nucleotide variations in at least two separate sites and a kit for conducting such method.

DESCRIPTION OF THE RELATED ART Understanding patterns of genetic variation is essential for studies of the genetic basis underlying the development of diseases and evolutionary history. Genetic variation can be assayed in various ways, each of which conveys different levels of information. It has been demonstrated that haplotypes of genetic markers {e.g., single nucleotide polymorphisms: SNPs) are more informative than genotypes (Sarah J. L., et al, Nucleic Acids Res. 33(18):el52(2005); Douglas, J.A., et al., Nature Genet, 28:361(2001); Martin E.R., et al., Am. J. Hum. Genet, 67:383(2000); Clark, A.G., et al., Genet. Epidemiol., 27:321(2004)).

Haplotyping is to determine which alleles lie on each of the two homologous chromosomes. For instance, an individual may have the genotype AB/ab (heterozygous at each of loci A and B), but could carry haplotypes AB and ab, or conversely, Ab and aB (Bernard A. K., et al., Nucleic Acids Res. 35(l):e6(2007)). Quantitative traits such as drug responsiveness and disease susceptibility may be more strongly correlated with certain haplotypes than with certain genotypes, particularly where several polymorphic loci fall within a single gene. Traditionally, haplotypes have been inferred by genotyping several generations of pedigree and tracing the segregation of markers.

In recent, a number of advanced methods have been proposed to determine haplotypes of genes. For examples, such approaches include PCR-RFLP (polymerase ^• chain reaction-restriction fragment length polymorphism) [5], allele specific oligonucleotide (ASO) probe, single-strand conformation polymorphism (SSCP) [10], primer extension [11], oligonucleotide ligation assay (OLA), heteroduplex anaysis [9] and using the multiplex amplification refractory mutation system (ARMS) [6,7]. U.S. Pat. No. 7,041,447 discloses haplotyping method for multiple distal nucleotide polymorphisms. This method involves the use of PCR (polymerase chain reaction) amplification and DNA ligation to bring the nucleotide polymorphisms on a particular allele of the gene into close proximity to facilitate the determination of haplotype structure. In addition, U.S. Pat. No. 6,844,154 discloses highthroughput methods for haplotyping based on hybridization, fluorescence detection, primer extension, MALDI-TOF and HPLC.

However, most of haplotyping methods suggested up to now are generally complicated, cost- and time-consuming. To make matters worse, there remains the generation of false results to be solved in these methods. Apolipoprotein (apoE) polymorphism is associated with the risk and the time of onset of Alzheimer's disease and the risk of developing cardiovascular disease. Therefore, much interest in apoE genotyping has been focused both for epidemiological research and for the purpose of diagnosing dyslipoproteinemia or dementia [1, 2]. ApoE is a major lipid transporter in human body and consists of 299 amino acids [3]. The three common isoforms of apoE are E2, E3, and E4 and they are encoded by the APOE-ε2, ε3, and ε4 genes, respectively. The isoforms differ only by a single amino acid at either position 112 and/or 158; E2 (Cysll2, Cys 158), E3 (Cysll2, Argl58) and E4 (Argll2, Argl58) [4]. As each individual possesses two allelic copies of a gene, the three apoE alleles can, thus, be combined into six different genotypes: E2/E2, E2/E3, E2/E4, E3/E3, E3/E4 and E4/E4.

The apoE alleles (ε2, ε3, and ε4) that encode the different isoforms can be distinguished by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) [5], allele specific oligonucleotide (ASO) probe, single-strand conformation polymorphism (SSCP) [aa], primer extension [aaa], oligonucleotide ligation assay (OLA), heteroduplex anaysis [a] and using the multiplex amplification refractory mutation system (ARMS) [6,7]. A common feature for the aforementioned method is that they are generally expensive and time-consuming.

Throughout this application, various patents and publications are referenced, and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive researches to develop a novel approach for haplotyping a nucleic acid molecule in more convenient and accurate manner. As a result, we have discovered that haplotyping could be successfully performed according to multiplex amplifications using at least six primers without false results.

Accordingly, it is an object of this invention to provide a method for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites.

It is another object of this invention to provide a kit for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 represents schematically the process and strategy of the present haplotyping method. A-NVS, a first site-present nucleotide variation specific primer 1; a-NVS, a first site-present nucleotide variation specific primer 2; B-NVS, a second site- present nucleotide variation specific primer 1; b-NVS, a second site-present nucleotide variation specific primer 2; TSPl, a target specific primer 1; and TSP2, a target specific primer 2. The asterisks indicate the position of polymorphic sites in a gene. In the haplotyping multiplex PCR, six different sizes of products (#l-#6) can be made from the combinatory working of these 6 primers. Rg. 2 represents schematic presentation of the difference between genotypes and haplotypes. Ten haplotypes result in different size patterns of multiplex PCR products according to the present method. A and B denote alleles on autosome. A is changed to a and B to b, giving rise to ten haplotypes.

Fig. 3 represents schematic illustration of the template and multiplex PCR primers for apoE genotyping analysis. The asterisk indicates the position of codon 112 (T to C) and codon 158 (C to T) polymorphic sites in apoE. The primers are described in Table 1. Two primers (APOE/Df, APOE/Dr) serve as an internal control for the quality of the PCR amplification and as template for the subsequent allele specific amplification. Four primers are designed for allele specific amplification. Fig. 4 represents the nucleotide sequences of the template and multiplex PCR primers for apoE haplotyping and genotyping analysis. The position of codon 112 (T to C) and codon 158 (C to T) polymorphic sites in apoE are indicated.

Fig. 5 represents the alleles of APOE. E2, E3 and E4 alleles exist, but there in no allele for 112C/158T in human. So there is no difference between 6 haplotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4 and E4/E4) and 6 genotypes. The allele specific products from multiplex PCR as in Figures 3 and 4 are represented.

Fig. 6 shows amplification products of the six common apoE haplotypes with hexaplex PCR. Characteristic band patterns are discernible for all six combination E2, E3, and E4 alleles. The amplicons were found to run at apparent molecular weight weights of: E2 (376 and 517 bp), E3 (311 and 376 bp) and E4 (311 and 447 bp). M, lOObp ladder; lane 1, E2/E2; lane 2, E2/E3; lane 3, E2/E4; lane 4, E3/E3; lane 5, E3/E4; and lane 6; E4/E4. Rg. 7 represents the results of the application of the present method to clinical samples for apoE haplotyping. Lanes 1 to 63, the band patterns generated after electrophoresis on an ethidium bromide-agarose gel can be translated into the different apoE haplotypes.

DETAILED DESCRIPTION OF THIS INVETNION

In one aspect of this invention, there is provided a method for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites, which comprises the steps of:

(a) annealing simultaneously primers of the following (i)-(iv) to the DNA molecule as a template;

(i) a first site-present nucleotide variation specific primer set (A-NVS and a-NVS) for amplifying nucleotide variations in a first site comprising (i-1) a first site-present nucleotide variation specific primer 1 (A-NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the first site and (i-2) a first site-present nucleotide variation specific primer 2 (a-

NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the first site,

(ii) a second site-present nucleotide variation specific primer set (B-NVS and b-NVS) for amplifying nucleotide variations in a second site comprising (ii-1) a second site-present nucleotide variation specific primer 1 (B-NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the second site and (ii-2) a second site-present nucleotide variation specific primer 2 (b-NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the second site, (iii) a target specific primer 1 (TSPl) comprising a nucleotide sequence hybridizable with a region located at upstream of the first site, and (iv) a target specific primer 2 (TSP2) comprising a nucleotide sequence hybridizable with a region located at downstream of the second site; wherein the primers are designed to produce six amplified products with different sizes from each other,

(b) performing at least two cycles of primer annealing, primer extending and denaturing using the primers to amplify the DNA molecule; and (c) determining the haplotype of the DNA molecule by analyzing the sizes of the amplified products of the step (b).

In another aspect of this invention, there is provided a kit for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites, which comprises (i) a first site-present nucleotide variation specific primer set (A-NVS and a-NVS) for amplifying nucleotide variations in a first site comprising (i-1) a first site-present nucleotide variation specific primer 1 (A-NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the first site and (i-2) a first site-present nucleotide variation specific primer 2 (a-NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the first site, (ii) a second site-present nucleotide variation specific primer set (B-NVS and b-NVS) for amplifying nucleotide variations in a second site comprising (ii-1) a second site- present nucleotide variation specific primer 1 (B-NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the second site and (ii-2) a second site-present nucleotide variation specific primer 2 (b-NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the second site, (iii) a target specific primer 1 (TSPl) comprising a nucleotide sequence hybridizable with a region located at upstream of the first site, and (iv) a target specific primer 2 (TSP2) comprising a nucleotide sequence hybridizable with a region located at downstream of the second site; wherein the primers are designed to produce six amplified products with different sizes from each other.

The present inventors have made intensive researches to develop a novel approach for haplotyping a nucleic acid molecule in more convenient and accurate manner. As a result, we have discovered that haplotyping could be successfully performed according to multiplex amplifications using at least six primers without false results. According to the present invention, the determination of haplotypes is easily made based on sizes of finally amplified products.

The present method is directed to the identification of at least two nucleotide variations in at least two separate sites by a single amplification reaction, thereby permitting to determine haplotypes a DNA molecule, e.g, a gene.

Up to now, it has not been practically proposed to simultaneously detect or identify two or more nucleotide variations in at least two separate loci by amplification reactions {e.g., PCR). The present invention provides for the first time a novel approach to simultaneously identify at least two nucleotide variations in at least two separate loci by a single amplification, thereby allowing for determining haplotypes of

DNA molecules in more convenient and accurate manner, which is due to primers having a unique structure and intriguing amplification strategies.

The term "haplotype" used herein means a 5' to 3' sequence of nucleotides found at one or more polymorphic sites (preferably, at least two polymorphic sites) in a locus on a single chromosome from an individual. The term "genotype" used herein means a 5' to 3' sequence of nucleotide pair(s) found at one or more polymorphic sites in a locus on a pair of homologous chromosome in an individual.

The term "nucleotide variation" used herein refers to a nucleotide polymorphism in a DNA sequence at a particular location among contiguous DNA segments that are otherwise similar in sequence. Such contiguous DNA segments include a gene or any other portion of a chromosome. For example, the nucleotide variation detected in the present invention includes deletion, insertion and substitution. Preferably, the nucleotide variation detected in this invention is a base substitution, more preferably, SNP (single nucleotide polymorphism). The present invention will be described in more detail with referring to Hg. 1 as follows:

The present method utilizes at least six primers. The number of primers is determined depending on the number of nucleotide variations to be analyzed. For determining the haplotype of a DNA molecule comprising nucleotide variations in two separate sites, the present method may be conducted using six primers: A-NVS, a-

NVS, A-NVS, b-NVS, TSPl and TSP2.

The first site-present nucleotide variation specific primer set, i.e., A-NVS and a- NVS primers are used to amplify a nucleotide variation in the first site. The second site-present nucleotide variation specific primer set, i.e., B-NVS and b-NVS primers are used to amplify a nucleotide variation in the second site.

The A-NVS and a-NVS primers have a nucleotide sequence hybridizable with the first nucleotide variation in the first site and a nucleotide sequence hybridizable with a second nucleotide variation in the first site, respectively. The B-NVS and b-NVS primers have a nucleotide sequence hybridizable with the first nucleotide variation in the second site and a nucleotide sequence hybridizable with a second nucleotide variation in the second site, respectively.

The TSPl and TSP2 primers are designed to be annealed to outermost regions of DNA molecules for giving the largest amplified products that serve as internal control in amplification reactions. As represented in Fig. 1, the six primers produce six amplicons with different sizes which enables haplotypes of genes to be determined by simply observing or analyzing sizes of amplicons produced.

According to a preferred embodiment, the first site-present nucleotide variation specific primer 1 (A-NVS) produces amplified products with either the target specific primer 2 (TSP2) or the second site-present nucleotide variation specific primer 2 (b- NVS).

According to a preferred embodiment, the first site-present nucleotide variation specific primer 2 (a-NVS) produces amplified products with the target specific primer 1 (TSPl).

According to a preferred embodiment, the second site-present nucleotide variation specific primer 1 (B-NVS) produces amplified products with the target specific primer 2 (TSP2). According to a preferred embodiment, the second site-present nucleotide variation specific primer 2 (b-NVS) produces amplified products with the target specific primer 1 (TSPl) or the first site-present nucleotide variation specific primer 1 (A-NVS).

According to a preferred embodiment, the target specific primer 1 (TSPl) produces amplified products (largest amplicons) with the target specific primer 2 (TSP2).

According to a preferred embodiment, the primers used in this invention, particularly, nucleotide variation specific primers have a unique structure or formula called as a dual specificity oligonucleotide structure. This dual specificity oligonucleotide (DSO) structure was first proposed by the present inventor (see WO 2006/095981) and then its nomenclature was changed to a dual priming oligonucleotide (DPO) structure.

The DPO embodies a novel concept in which its hybridization or annealing is dually determined by the 5'-high T_m specificity portion (or the 5'-first priming portion) and the 3'-low T_m specificity portion (or the 3'-second priming portion) separated by the separation portion, exhibiting dramatically enhanced specificity (see WO 2006/095981). As such, the DPO has eventually two primer segments with distinct annealing properties: the 5'-first priming portion that initiates stable priming, and the 3'-second priming portion that determines target-specific extension.

More preferably, the A-NVS, a-NVS, B-NVS and/or b-NVS primers have the dual priming oligonucleotide structure represented by the following general formula I:

5'-Ap-Y_q-V_r-3' (I) wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, V_r represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and A, Y, and V are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the variation adjacent specificity portion from the variation specificity portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the variation adjacent specificity portion and the variation specificity portion such that the overall annealing specificity of the primers is enhanced.

The separation portion comprising at least three universal bases delineates the boundary between the variation adjacent specificity portion and the variation specificity portion, resulting in separation of the variation adjacent specificity portion from the variation specificity portion in view of annealing events. Such separation permits the annealing specificity and priming of the primers to be determined dually by the variation adjacent specificity portion and the variation specificity portion, finally dramatically increasing the overall annealing specificity of the primers.

According to a preferred embodiment, the universal base in the separation portion is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'- deoxyinosine, 2-aza-2'-deoxyinosine, 2'-OMe inosine, 2'-F inosine, deoxy 3- nitropyrrole, 3-nitropyrrole, 2'-0Me 3-nitropyrrole, 2'-F 3-nitropyrrole, l-(2'-deoxy- beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole, 5-nitroindole, 2'-0Me 5- nitroindole, 2'-F 5-nitroindole, deoxy 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4-aminobenzimidazole, deoxy nebularine, 2'-F nebularine, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3-nitropyrrole, morpholino-5-nitroindole, morpholino- nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3- nitropyrrole, phosphoramidate-5-nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4- nitrobenzimidazole, phosphoramidate- 3-nitropyrrole, 2'-0-methoxyethyl inosine, 2'0-methoxyethyl nebularine, 2'-O- methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro-benzimidazole, 2'-O- methoxyethyl 3-nitropyrrole, and combinations thereof. More preferably, the universal base or non-discriminatory base analog is deoxyinosine, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropyrrole or 5-nitroindole, most preferably, deoxyinosine. It is preferable that the separation portion comprises contiguous nucleotides having at least three universal bases, preferably, deoxyinosine.

Preferably, the variation adjacent specificity portion is longer than the variation specificity portion. The variation adjacent specificity portion is preferably 15-40 nucleotides, more preferably 15-25 nucleotides in length. It is preferable that the variation specificity portion is 3-15 nucleotides, more preferably 6-13 nucleotides in length.

The separation portion is preferably 3-10 nucleotides, more preferably 4-8 nucleotides, most preferably 5-7 nucleotides in length.

According to a preferred embodiment, the T_m of the variation adjacent specificity portion ranges from 40⁰C to 80⁰C, more preferably 45°C to 65°C. The T_m of the variation specificity portion ranges preferably from 10⁰C to 40⁰C. It is preferable that the T_m of the separation portion ranges from 3°C to 15°C.

The variation specificity portion of primers has a nucleotide complementary or corresponding to the nucleotide variation. Where the primers are annealed to the sense strand of the target DNA molecule, the variation specificity portion has a nucleotide complementary to the nucleotide variation. In contrast, where the primers are annealed to the antisense strand of the target DNA molecule, the variation specificity portion has a nucleotide corresponding to the nucleotide variation.

According to a preferred embodiment, the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at the 3'-end of the variation specificity portion or at 1-10 nucleotides apart from the 3'-end of the variation specificity portion. More preferably, the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at 2-7 nucleotides, more still preferably, 3-6 nucleotides apart from the 3'-end of the variation specificity portion. Most preferably, the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at the center or around the center of the variation specificity portion. For instance, where the variation specificity portion is 8 nucleotides in length, the nucleotide complementary or corresponding to the nucleotide variation is located at 3- 6 nucleotides, preferably 4-5 nucleotides, more preferably 4 nucleotides apart from the 3'-end of the variation specificity portion.

Such positioning of the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion has some advantages.

For example, according to the conventional methods for detecting SNP using the conventional primers, the nucleotide variation-specific base is located at the 3'-end of primers. This position is responsible for the slight difference in T_m values of annealing occurrence and non-annealing occurrence {i.e., mismatching) of the nucleotide variation-specific base. Thus, when mismatching events occur, amplification reactions are very likely to occur, giving rise to the generation of false results. Conversely, where the nucleotide variation-specific base is located around at the center of conventional primers, the difference in T_m values between annealing and non-annealing {i.e., mismatching) becomes larger. However, most of thermostable polymerases used in amplification reactions catalyze the reactions in disregard of mismatching in the central region of primers, resulting in the generation of fals- positive results. Surprisingly, the present invention ensures to completely overcome the shortcoming of conventional techniques described above. For illustration, where the nucleotide variation-specific base located around at the center of the variation specificity portion of the present primers is mismatched, the T_m value of the variation specificity portion becomes much lower because the mismatching occurs at the center of the variation specificity portion. However, considering the overall structure of the present primers, the mismatching event is recognized to occur around the 3'-end of primers and therefore thermostable polymerases do not catalyze reactions. Therefore, mismatching of the nucleotide variation-specific base induces no false-positive results.

The nucleotide variation specific primers described above have overlapping sequence to each other. That is, A-NVS and a-NVS primers annealed to the first polymorphic site, or B-NVS and b-NVS primers annealed to the second polymorphic site have overlapping sequence to each other. Generally, where primers having overlapping sequence are used in the same amplification reaction, a duplex between primers are formed to result in the generation of false amplification results. However, the nucleotide variation specific primers used in the present method do not form duplex structures although they carry overlapping sequences. The utilization of overlapping sequences in the nucleotide variation specific primers enables the present method to be performed according to the proposed amplification strategy.

The TSPl and TSP2 primers used in the present invention may have any structure or formula so long as they are annealed to outermost regions of DNA molecules for giving the largest amplified products that serve as internal control in amplification reactions.

According to a preferred embodiment, the TSPl and/or TSP2 primers have a dual priming oligonucleotide structure represented by the following general formula II:

5'-Xp-Y_q-Z_r-3' (II) wherein X_p represents a 5'-first priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, Z_r represents a 3'-second priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; T_m of the 5'-first priming portion is higher than that of the 3'-second priming portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the 5'-first priming portion from the 3'-second priming portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the 5'-first priming portion and the 3'-second priming portion such that the overall annealing specificity of the primers is enhanced. The preferable structure of the TSPl and TSP2 primers (i.e., dual priming oligonucleotide structure) is fundamentally identical to that of the A-NVS, a-NVS, B- NVS and b-NVS primers. Therefore, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification. According to the most preferred embodiment, the A-NVS, B-NVS, A-NVS and/or

B-NVS primers have the structure represented by the following general formula III:

5'-A_p-CdIVVrS¹ (HI) wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the template DNA molecule, (dl)_q represents a separation portion comprising contiguous deoxyinosine bases, V₁- represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the template DNA molecule, p is an integer of 15-25, q is an integer of 4-8 and r is an integer of 6-13, and A and V are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_n, in the three portions; the separation portion separates the variation adjacent specificity portion from the variation specificity portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the variation adjacent specificity portion and the variation specificity portion such that the overall annealing specificity of the primers is enhanced. According to a preferred embodiment, the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion {i.e., the nucleotide variation-specific base is located at the center or around the center of the variation specificity portion (V_r). For instance, where V₁- is 6, 7, 8, 9, 10, 11, 12 and 13 nucleotides in length, the nucleotide variation-specific base is located 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7 and 6-8 nucleotides apart from the 3'-end, respectively.

The term "primer" as used herein refers to an oligonucleotide, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand (template) is induced, i.e., in the presence of nucleotides and an agent for polymerization, such as DNA polymerase, and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer of this invention can be comprised of naturally occurring dNMP {i.e., dAMP, dGM, dCMP and dTMP), modified nucleotide, or non-natural nucleotide. The primer can also include ribonucleotides. For example, the primer used in this invention may include nucleotides with backbone modifications such as peptide nucleic acid (PNA) (M. Egholm et al., Nature, 365:566-568(1993)), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methyl phosphonate DNA, nucleotides with sugar modifications such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2'-0-allyl DNA, 2'-0-alkynyl DNA, hexose DNA, pyranosyl RNA, and anhydrohexitol DNA, and nucleotides having base modifications such as C-5 substituted pyrimidines (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethynyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyridyl-), 7-deazapurines with C-7 substituents (substituents including fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-), inosine, and diaminopurine. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact length of the primers will depend on many factors, including temperature, application, and source of primer. The term "annealing" or "priming" as used herein refers to the apposition of an oligodeoxynucleotide or nucleic acid to a template nucleic acid, whereby the apposition enables the polymerase to polymerize nucleotides into a nucleic acid molecule which is complementary to the template nucleic acid or a portion thereof. The term used "hybridizing" used herein refers to the formation of a double- stranded nucleic acid from complementary single stranded nucleic acids. There is no intended distinction between the terms "annealing" and "hybridizing", and these terms will be used interchangeably. The sequences of the primers may comprise some mismatches, so long as they can be hybridized with templates and serve as primers. The variation adjacent specificity portion and the variation specificity portion are designed to have a nucleotide sequence substantially complementary to the template DNA molecule. The term "substantially complementary" is used herein to mean that the primer is sufficiently complementary to hybridize selectively to a template nucleic acid sequence under the designated annealing conditions or stringent conditions, such that the annealed primer can be extended by a polymerase to form a complementary copy of the template. Most preferably, the variation adjacent specificity portion and the variation specificity portion have a nucleotide sequence perfectly complementary to template DNA molecules, i.e., no mismatches.

The process for amplifying the DNA molecule by primer annealing, primer extending and denaturing is well known to those of skill in the art. Suitable annealing or hybridization conditions may be routinely determined by optimization procedures. Conditions such as temperature, concentration of components, hybridization and washing times, buffer components, and their pH and ionic strength may be varied depending on various factors, including the length and GC content of primer and target nucleotide sequence. The detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. N.Y.(1999).

According to a preferred embodiment, the annealing is performed at temperature of 45-72°C, more preferably, 50-70⁰C, most preferably 60-68⁰C.

The present method can determine haplotypes of any DNA molecule, e.g., a gene or any other portion of a chromosome.

The DNA molecule may be either gDNA (genomic DNA) or cDNA (complementary DNA). Such molecule may be either DNA or RNA. The molecule may be in either a double-stranded or single-stranded form. Where the nucleic acid as starting material is double-stranded, it is preferred to render the two strands into a single-stranded or partially single-stranded form. Methods known to separate strands includes, but not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action), and binding proteins. For instance, strand separation can be achieved by heating at temperature ranging from 80⁰C to 105⁰C. General methods for accomplishing this treatment are provided by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001).

Where a mRNA is employed as starting material, a reverse transcription step is necessary prior to performing annealing step, details of which are found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988)). For reverse transcription, an oligonucleotide dT primer hybridizable to poly A tail of mRNA is used. The oligonucleotide dT primer is comprised of dTMPs, one or more of which may be replaced with other dNMPs so long as the dT primer can serve as primer. Reverse transcription can be done with reverse transcriptase that has RNase H activity. If one uses an enzyme having RNase H activity, it may be possible to omit a separate RNase H digestion step by carefully choosing the reaction conditions.

The present methods do not require that the template nucleic acid molecules have any particular sequence or length. The primers used for the present invention is hybridized or annealed to a site on the template such that double-stranded structure is formed. Conditions of nucleic acid annealing suitable for forming such double stranded structures are described by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).

A variety of DNA polymerases can be used in the extension step of the present methods, which includes "Klenow" fragment of E coli DNA polymerase I, a thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase which may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis ffavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). When a polymerization reaction is being conducted, it is preferable to provide the components required for such reaction in excess in the reaction vessel. Excess in reference to components of the extension reaction refers to an amount of each component such that the ability to achieve the desired extension is not substantially limited by the concentration of that component. It is desirable to provide to the reaction mixture an amount of required cofactors such as Mg²⁺, dATP, dCTP, dGTP, and dTTP in sufficient quantity to support the degree of the extension desired.

Annealing or hybridization in the present method is performed under stringent conditions that allow for specific binding between the primer and the template nucleic acid. Such stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters. In the present method, the annealing step is generally performed under high stringent conditions.

In the most preferable embodiment, the amplification is performed in accordance with PCR (polymerase chain reaction) which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159.

Fundamentally, the present method follows multiplexing amplification using several primers annealed to several target sequences. Preferably, the amplification is performed in accordance with multiplex PCR. According to a conventional multiplex PCR, the results obtained with multiplex

PCR are frequently complicated by the artifacts of the amplification procedure. These include "false-negative" results due to reaction failure and "false-positive" results such as the amplification of spurious products, which may be caused by annealing of the primers to sequences which are related to but distinct from the true recognition sequences. Therefore, elaborate optimization steps of multiplex PCR are conducted to reduce such false results; however, the optimization of the reaction conditions for multiplex PCR may become labor-intensive and time-consuming and unsuccessful. The present method amplifies simultaneous a variety of target sequence for haplotyping with no false results in a single PCR reaction to completely overcome shortcomings associated with conventional multiplex PCR.

The analysis of amplified products in the present invention may be conducted by various methods or protocols. For example, the amplified products can be analyzed by electrophoresis on a suitable gel {e.g., agarose gel). The amplified products could be also detected on a denaturing polyacrylamide gel by autoradiography or nonradioactive detection methods, such as silver staining (Gottschlich et al_v (1997) Res. Commun. MoI. Path. Pharm. 97, 237-240; Kociok, N., et al. (1998) MoI. Biotechnol. 9, 25-33), or by using fluoresenscent-labelled oligonucleotides (Bauer D., et al., (1993) Nucleic Acids Res. 21, 4272-4280; Ito, T. et al., (1994) FEBS Lett. 351, 231-236. Luehrsen, K.R. et al., (1997) BioTechniques 22, 168-174; Smith, N. R. et al., (1997) BioTechniques 23, 274-279), and the use of biotinylated primers (Korn, B. et al., (1992) Hum. MoI. Genet 1, 235-242; Tagle, D.A. et al., (1993) Nature 361. 751-753; Rosok, O. et al., (1996) BioTechniques 21, 114-121). The primers used in the present invention are designed to produce amplicons with different sizes for identifying nucleotide variations; therefore the simple analysis or observation of size difference of amplified products enables to haplotype DNA molecules in more convenient manner.

The present method aforementioned is described with referring to a process using six primers for producing six amplicons. However, it will be obvious to one of skill in the art that the present method can be performed using further primers for identifying further nucleotide variations on the same DNA molecule.

The DNA molecule, e.g., gene, haplotyped by the present invention is not limited. Preferably, the genes of which haplotypes are determined by the present invention include those for which haplotypes have been shown to be relevant with the development of diseases and the response to drugs in human. For example, such important gene includes genes encoding apolipoprotein E (apoE), thiopurine S- methyltransferase (TPMT), β2 receptor, OPRMl and interleukin-4 (IL-4) receptor α.

According to a preferred embodiment of the present kit, the A-NVS, a-NVS, B-

NVS and/or b-NVS primers have a dual priming oligonucleotide structure represented by the following general formula I:

5'-A_p-Y_q-V_r-3' (I) wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, V₁- represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the variation adjacent specificity portion from the variation specificity portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the variation adjacent specificity portion and the variation specificity portion such that the overall annealing specificity of the primers is enhanced. According to a preferred embodiment of the present kit, the TSPl and/or TSP2 primers have a dual priming oligonucleotide structure represented by the following general formula II:

5'-X_p-Y_q-Z_r-3' (II) wherein X_p represents a 5'-first priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, Z₁- represents a 3'-second priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; T_m of the 5'-first priming portion is higher than that of the 3'-second priming portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the 5'-first priming portion from the 3'-second priming portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the 5'-first priming portion and the 3'-second priming portion such that the overall annealing specificity of the primers is enhanced.

Since, the primers contained in the present kit are identical to those used in the present haplotyping method, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

The present kit may optionally include the reagents required for performing

PCR reactions such as buffers, thermostable DNA polymerase, DNA polymerase cofactors, and deoxyribonudeotide-5-triphosphates. Optionally, the kit may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. The kits may also include reagents necessary for performing positive and negative control reactions.

Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the current disclosure. The kits, typically, are adapted to contain in separate packaging or compartments the constituents afore- described.

The features and advantages of this invention are will be summarized as follows:

(a) the present invention is carried out in accordance with multiplex amplification using at least six primers;

(b) the present invention enables to simultaneously identify at least two types of nucleotide variations in at least two separate sites in higher fidelity, contributing to haplotyping of a DNA molecule in a single multiplex amplification;

(c) the present haplotyping method is carried out in accordance with relatively simple and convenient process such as multiplex amplification and analysis of amplicon sizes;

(d) according to the present invention, the determination of haplotypes of genes is readily made by a simple analysis of sizes of amplification products; (e) interestingly, it is possible to simultaneously obtain haplotypes of genes and genotypes of genes in a single multiplex amplification by the present invention; and

(f) even though the present invention is carried out in a relatively simple fashion, the results for haplotyping of genes are shown to be much more accurate than any conventional methods.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLE Material and Methods

DNA isolation Genomic DNA was isolated from 200 μl aliguots of human whole blood samples using spin columns (Bioneer AccuPrep^® Genomic DNA extraction kit, Korea) according to the manufacturer's recommendations. For quality control, known controls representing all polymorphic variant were included.

Quantification of genomic DNA

Quantification of DNA was performed using a NanoDrop^® ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA).

DPO primer design We contemplated and designed scheme for determining haplotypes of the apoE alleles at two SNP loci by use of six primers (Rg. 3). Four primers were prepared to be hybridized with two SNP sites and two primers were prepared to be hybridized with the outer region. The total six primers were also designed to produce amplicons (six different amplification products) with different sizes, which permits to haplotype the apoE gene based on PCR product sizes. A 779 bp was always obtained as control for the success of amplification using the APOE/Df and APOE/Dr (Table 1). TABLE 1

Name³ Direction Sequence(5^3") ^b Tm(TC)

APOE/Df

Forward CGGCCTCCTAGCTCCTTαilllCrCTGCCr 78.0 (SEQ ID NO:1)

APOE/Dr

Reverse CTGCAGGCGTATCTGCΠΠHTGCΓCCTC 69.3 (SEQ ID NO:2)

APOE/Df-al^c

Forward GGCGCGGACATGIIIIICGTGCGCG 84.3 (SEQ ID NO:3)

APOE/Dr-al^c

Reverse CGGTACTGCACCAIIIIICCGCACA 67.1 (SEQ ID NO:4)

AP0E/Df-a2^c

Forward GCGATGCCGATGACIIIIIGAAGCGCC 80.8 (SEQ ID NO:5)

APOE/Dr-a2^c

Reverse GCCCCGGCCTGGTACAIIIIIAGGCACTT 72.5 (SEQ ID NO:6) a Df and Dr represent DPO forward primer and DPO reverse primer, respectively. b The position of a single base variation in apoE-specif ic primers is highlighted in gray. c al and a2 represent allele 1 (codon 112)-specific primer and allele 2 (codon 158)-specific primer, respectively.

The symbol "I" denotes deoxyinosine.

HexaplexPCR

To assess the identified haplotypes of the aopE gene using hexaplex PCR, specific primers were utilized (Table 1). The total 20 μl PCR reaction contained 20-30 ng genomic DNA, 4 μl of 5 X primer mixture (final 0.25 μM each), 10 μl of 2 X Master Mix (Seegene, Korea) containing 5% DMSO. The cycling conditions were as follows: denaturation for 5 min at 94⁰C; amplification for 35 cycles, with denaturation for 30 sec at 94⁰C, annealing for 30 sec at 65⁰C and extension for 1 min at 72⁰C. The amplified PCR products were separated on 2% agarose gel stained with ethidium bromide. Sequencing

The target PCR products on the agarose gel were purified for sequencing using the Gel DNA recovery kit (ZYMO RESEΞARCH). The purified PCR products were sequenced in ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Clinical Application

Genomic DNA samples were obtained from sixty-three patients not being analyzed for apo E haplotypes. Hexaplex PCR amplifications were conducted using 20 ng of genomic DNA samples as described above. For confirmation, sequencing was carried out as described hereinabove.

Results

The amplified sequence of the apoE gene encompasses nucleotide substitutions that result in the arginine-cystein interchange at positions 112 and 158. Figure 6 shows the band patterns obtained from the analysis of apoE haplotypes. Each apoE haplotype was readily recognizable based on sizes of PCR products and there was no ambiguity between them. A common band of 779 bp size as the internal control appeared in all the cases, and PCR analysis gave the expected products (Fig. 6). The haplotyping results by hexaplex PCR were completely consistent with those by DNA sequence. The results are summarized in Table 2. TABLE 2

5 E3/E4 112C(F), 112T(R), 158C(F) Heterozygote 311+376+447

6 E4/E4 112C(F), 158C(F) Homozygote 311+447 a haplotypes are defined by the presence of two polymorphi loci at codon 112 and 158 of the apoE gene. b band patterns obtained from the analysis of apoE genotypes. c Internal control, A 779 bp was always obtained as control for the success of amplification.

The present haplotyping method was conducted using genomic samples from sixty-three patients not being analyzed for apoE haplotypes. The results were shown as band patterns in Fig. 7. The haplotyping results by hexaplex PCR were complicatedly consistent with those by DNA sequence. The results are summarized in Table 3. TABLE 3

haplotypes and genotypes determined by the present method.

Discussion

Now, a number of methods are available for SNP genotyping and haplotyping, and the choice of the method depends on the assay scale. In a large-scale assay, an allele-specific hybridization method using TaqMan probe, a fluorescence resonance energy transfer probe, or a molecular beacon is most commonly used for SNP genotyping. This method allows accurate allelic discrimination in a one-step procedure without separation of the analytes or removal of the surplus fluorescent contaminants. On the other hand, the genotyping methods for small-scale assay require complicated processes and a skillful analyst; many of these methods are gel electrophoresis-based techniques, such as single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis, restriction isotyping and conformation-sensitive gel electrophoresis. We have established a new haplotyping method for the detection of the human apoE condon 112 and 158 SNPs. The haplotyping method of the present invention using DPO primers shows accurate results in not only small scale assay but also assay for determining multiple polymorphisms.

Haplotypes are specific combination of genetic variants located on an allele. In some studies, haplotypes conferring significantly to the risk for a complex disorder could be assigned while a single casual variant eluded definitive identification. The commonly used haplotying methods are laborious, expensive, time-consuming, or prone to errors. Therefore, many studies have limitations in the haplotype determination. In contrast, the present haplotyping method by multiplex SNP PCR amplification allows for the haplotyping of multiple SNPs {e.g., two SNPs) by one PCR reaction. As described in Fig. 2 for illustration, where two SNPs exist on an allele, ten haplotypes are theoretical possible. In such case, the present haplotyping method permits to identify the seven haplotypes by one multiplex PCR reaction.

The new haplotyping strategy that we propose is based on DPO system with multiplex SNP PCR. These include not only faster analysis and the standardization of both amplification and detection steps, but also the practice of a very elegant approach, no prone to mistakes.

References

1. Corder, E.H., Saunders,A.M., Strittmatter, WJ., Schmechel,D.E.,Gaskell,P.C, Small,G.W.,Roses, A.D., HainesJ.L and Pericak-Vance,M.A (1993) Gene dose of apolipoprotein E tye allele and the risk of Alzheimer's disease in late onset families.Science,261,921-923

2. Wilson,P.W., Schaefer,E.l, Larson,M.G. and Ordovas,J.M.(1996) Apolipoprotein E alleles and risk of coronary disease. A meta-analysis. Arterioscler. Thromb. Vase. Biol.

16, 1250-1255

3. R.W. Mahley, S.C. Rail, Apolipoprotein E. cholesterol transport protein with expanding role in cell biology, Science 240 (1988) 622-630.

4. A.M. Saunders, WJ. Strittmatter, D. Schmeched, RH. GeorgeHyslop, M.A. Perickak- Vance, S.H. Joo, BJ. Rosi, J. F. Gusella, D.R. Crapper-MacLachlan, MJ Alberts. Association of Apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease, Neurology 43 (1993) 467-472.

5. Zivelin A, Rosenberg N, Peretz H, Amit Y, Kombort N, Seligsohn U. Improved method for genotyping Apolipoprotein E polymorphisms by a PCR-based assay simultaneously utilizing two distinct restriction enzymes. Clin Chem 1997, 43(9); 1657- 1659

6. Wenham PR, Newton CR, Price WH. Analysis of Apolipoprotein E genotypes by the amplification refractory mutation system. Clin Chem 1991, 37(2); 241-244.

7. Donohoe GG, Salomaki A, Lehtimaki T, Pulkki K, Kairisto V : Rapid identification of Apolipoprotein E genotype by multiplex amplification refactory mutation system. Clin Chem 1999, 45(1);43-146.

8. J.Y. Chon, KJ. Kim, IT. Hwang, YJ. Kim, D.H. Lee, I.K. Lee and J.K Kim. Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.

9. BoIIa, M.K., Wood,N.and Humphries,S.E(1999) Rapid determination of apolipoprotein E genotype using a heteroduplex generator. J. Lipid Res., 40, 2340-

2345.

10. Tsai,M.Y, Suess,P., Schwichtenberg,K., EckfeldtJ.H.,YuanJ.,Tuchman,M.and Hunninghake,D.(1993) Determination of apolipoprotein E genotypes by single-strand conformational polymorphism. Clin. Chem., 39, 2121-2124

11. Syvanen,A.C, Aalto-Setala,K., Harju,L, Kontula,K. and Soderlund,H.(1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics,8,684-692.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A method for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites, which comprises the steps of:

(ii) a second site-present nucleotide variation specific primer set (B-NVS and b-NVS) for amplifying nucleotide variations in a second site comprising (M-I) a second site-present nucleotide variation specific primer 1 (B-NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the second site and (ii-2) a second site-present nucleotide variation specific primer 2 (b-NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the second site, (iii) a target specific primer 1 (TSPl) comprising a nucleotide sequence hybridizable with a region located at upstream of the first site, and (iv) a target specific primer 2 (TSP2) comprising a nucleotide sequence hybridizable with a region located at downstream of the second site; wherein the primers are designed to produce six amplified products with different sizes from each other,

(b) performing at least two cycles of primer annealing, primer extending and denaturing using the primers to amplify the DNA molecule; and

(c) determining the haplotype of the DNA molecule by analyzing the sizes of the amplified products of the step (b).

2. The method according to claim 1, wherein the first site-present nucleotide variation specific primer 1 (A-NVS) produces amplified products with either the target specific primer 2 (TSP2) or the second site-present nucleotide variation specific primer 2 (b-NVS).

3. The method according to claim 1, wherein the first site-present nucleotide variation specific primer 2 (a-NVS) produces amplified products with the target specific primer 1 (TSPl).

4. The method according to claim 1, wherein the second site-present nucleotide variation specific primer 1 (B-NVS) produces amplified products with the target specific primer 2 (TSP2).

5. The method according to claim 1, wherein the second site-present nucleotide variation specific primer 2 (b-NVS) produces amplified products with the target specific primer 1 (TSPl) or the first site-present nucleotide variation specific primer 1 (A-NVS).

6. The method according to claim 1, wherein the target specific primer 1 (TSPl) produces amplified products with the target specific primer 2 (TSP2).

7. The method according to claim 1, wherein the A-NVS, a-NVS, B-NVS and/or b-NVS primers have a dual priming oligonucleotide structure represented by the following general formula I:

5'-A_p-Y_q-V_r-3' (I) wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, V₁- represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and A, Y, and V are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the variation adjacent specificity portion from the variation specificity portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the variation adjacent specificity portion and the variation specificity portion such that the overall annealing specificity of the primers is enhanced.

8. The method according to claim 7, wherein the universal base is selected from the group consisting of deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 2-aza-2'- deoxyinosine, 2'-OMe inosine, 2'-F inosine, deoxy 3-nitropyrrole, 3-nitropyrrole, 2'-OMe 3-nitropyrrole, 2'-F 3-nitropyrrole, l-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, deoxy 5-nitroindole, 5-nitroindole, 2'-0Me 5-nitroindole, 2'-F 5-nitroindole, deoxy 4- nitrobenzimidazole, 4-nitrobenzimidazole, deoxy 4-aminobenzimidazole, 4- aminobenzimidazole, deoxy nebularine, 2'-F nebularine, 2'-F 4-nitrobenzimidazole, PNA-5-introindole, PNA-nebularine, PNA-inosine, PNA-4-nitrobenzimidazole, PNA-3- nitropyrrole, morpholino-5-nitroindole, morpholino-nebularine, morpholino-inosine, morpholino-4-nitrobenzimidazole, morpholino-3-nitropyrrole, phosphoramidate-5- nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate- 4- nitrobenzimidazole, phosphoramidate-3-nitropyrrole, 2'-0-methoxyethyl inosine, 2¹O- methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitrobenzimidazole, 2'-0-methoxyethyl 3-nitropyrrole, and combinations thereof.

9. The method according to claim 8, wherein the universal base is deoxyinosine, 1- (2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole or 5-nitroindole.

10. The method according to claim 9, wherein the universal base is deoxyinosine.

11. The method according to claim 7, wherein the separation portion comprises contiguous nucleotides having at least three universal bases.

12. The method according to claim 7, wherein the variation adjacent specificity portion is 15-40 nucleotides in length.

13. The method according to claim 7, wherein the variation specificity portion is 3-15 nucleotides in length.

14. The method according to claim I₁ wherein the separation portion is 3-10 nucleotides in length.

15. The method according to claim 7, wherein the T_m of the variation adjacent specificity portion ranges from 40⁰C to 80⁰C.

16. The method according to claim 7, wherein the T_m of the variation specificity portion ranges from 10⁰C to 40⁰C.

17. The method according to claim 7, wherein the T_m of the separation portion ranges from 3°C to 15°C.

18. The method according to claim 7, wherein the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at the 3'-end of the variation specificity portion or at 1-10 nucleotides apart from the 3'-end of the variation specificity portion.

19. The method according to claim 18, wherein the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at 2-7 nucleotides apart from the 3'-end of the variation specificity portion.

20. The method according to claim 19, wherein the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at 3-6 nucleotides apart from the 3'-end of the variation specificity portion.

21. The method according to claim 18, wherein the nucleotide complementary or corresponding to the nucleotide variation in the variation specificity portion is located at the center of the variation specificity portion.

22. The method according to claim 1, wherein the TSPl and/or TSP2 primers have a dual priming oligonucleotide structure represented by the following general formula II:

5'-Xp-Y_q-Z_r-3' (II) wherein X_p represents a 5'-first priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, Z_r represents a 3'-second priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; T_m of the 5'-first priming portion is higher than that of the 3'-second priming portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the 5'-first priming portion from the 3'-second priming portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the 5'-first priming portion and the 3'-second priming portion such that the overall annealing specificity of the primers is enhanced.

23. The method according to claim 1, wherein the nucleotide variation is SNP (single nucleotide polymorphism).

24. The method according to claim 1, wherein the step (b) is carried out by multiplex PCR (polymerase chain reaction).

25. A kit for determining the haplotype of a DNA molecule comprising nucleotide variations in at least two separate sites, which comprises (i) a first site-present nucleotide variation specific primer set (A-NVS and a-NVS) for amplifying nucleotide variations in a first site comprising (i-1) a first site-present nucleotide variation specific primer 1 (A-NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the first site and (i-2) a first site-present nucleotide variation specific primer 2 (a-NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the first site, (ii) a second site-present nucleotide variation specific primer set (B-NVS and b-NVS) for amplifying nucleotide variations in a second site comprising (ii-1) a second site-present nucleotide variation specific primer 1 (B- NVS) comprising a nucleotide sequence hybridizable with a first nucleotide variation in the second site and (i-2) a second site-present nucleotide variation specific primer 2 (b-NVS) comprising a nucleotide sequence hybridizable with a second nucleotide variation in the second site, (iii) a target specific primer 1 (TSPl) comprising a nucleotide sequence hybridizable with a region located at upstream of the first site, and (iv) a target specific primer 2 (TSP2) comprising a nucleotide sequence hybridizable with a region located at downstream of the second site; wherein the primers are designed to produce six amplified products with different sizes from each other.

26. The kit according to claim 25, wherein the A-NVS, a-NVS, B-NVS and/or b-NVS primers have a dual priming oligonucleotide structure represented by the following general formula I:

5'-A_p-Y_q-V_r-3' (I) wherein A_p represents a variation adjacent specificity portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, V_r represents a variation specificity portion having a nucleotide complementary or corresponding to the nucleotide variation and a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and A, Y, and V are deoxyribonucleotide or ribonucleotide; T_m of the variation adjacent specificity portion is higher than that of the variation specificity portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the variation adjacent specificity portion from the variation specificity portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the variation adjacent specificity portion and the variation specificity portion such that the overall annealing specificity of the primers is enhanced.

27. The kit according to claim 25, wherein the TSPl and/or TSP2 primers have a dual priming oligonucleotide structure represented by the following general formula II:

5'-X_p-Y_q-Z_r-3' (II) wherein X_p represents a 5'-first priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, Y_q represents a separation portion comprising at least three universal bases, Z_r represents a 3'-second priming portion having a nucleotide sequence substantially complementary to the template DNA molecule, p, q and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide; T_m of the 5'-first priming portion is higher than that of the 3'-second priming portion and the separation portion has the lowest T_m in the three portions; the separation portion separates the 5'-first priming portion from the 3'-second priming portion in terms of annealing events to the template DNA molecule, whereby the annealing specificity and priming of the primers are determined dually by the 5'-first priming portion and the 3'-second priming portion such that the overall annealing specificity of the primers is enhanced.