WO2004063373A1

WO2004063373A1 - Dna size markers and method for preparing them

Info

Publication number: WO2004063373A1
Application number: PCT/KR2003/000064
Authority: WO
Inventors: Jong-Yoon Chun
Original assignee: Seegene, Inc.
Priority date: 2003-01-13
Filing date: 2003-01-13
Publication date: 2004-07-29
Also published as: WO2004063322A3; WO2004063322A2; AU2003201778A1

Abstract

The present invention relates to a method for preparing DNA size markers by virtue of multiplex polymerase chain reaction and DNA size markers prepared thereby. The present method comprises the steps of: (a) selecting a plurality of specific regions on target DNA molecule(s) to be amplified so that each amplified product has a definite and desired length; (b) preparing a plurality of first primer pairs for amplifying the specific regions in which a 5'-end portion of each forward primer of the first primer pairs has a substantially common nucleotide sequence, a 5'-end portion of each reverse primer of the first primer pairs has a substantially common nucleotide sequence and a 3'-end portion of the first primer pairs has an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; (c) performing polymerase chain reaction of the specific regions using the first primer pairs so that DNA molecules with definite and desired lengths are amplified; and (d) performing multiplex polymerase chain reaction of the amplified products in the same reaction using one type of a third primer pair in which each third primer comprises a nucleotide sequence to be hybridized with a nucleotide sequence at each end portion of the amplified products.

Description

DNASIZE MARKERSAND METHOD FORPREPARINGTHEM

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for preparing DNA size markers and DNA size markers prepared thereby, more particularly, to a method for preparing DNA size markers by virtue of multiplex polymerase chain reaction and DNA size markers prepared thereby.

DESCRIPTION OF THE RELATED ART The DNA molecular weight markers are necessary to determine the molecular weight or the base pair length of nucleic acids. A large number of DNA marker fragments are available from numerous suppliers. These marker fragments are obtained either by restriction digests of several bacteriophage or plasmid DNAs, or polymerase chain reaction (PCR) amplification. However, there is a limitation in obtaining desired marker fragments from the methods of restriction digests (Carlson et al., U.S. Pat. No. 5,316,908). Thus, PCR amplification has been applied to obtain the desired DNA marker fragments (Amills et al., Genet. Anal, 13:147- 149(1996); and Dawson, U.S. Pat. No. 5,714,326). According to the above method, each marker ladder is amplified by each separate PCR reaction and then the PCR products are combined to be used as DNA marker ladders because the multiple PCR approach rendered a discontinuous ladder with many gaps and unspecific bands (Amills et al., Genet. Anal, 13:147-149 (1996); and Dawson, U.S. Pat. No. 5,714,326). However, such approach is in fact time consuming when compared with the multiplex PCR approach. No technique has been described, permitting the use of multiplex PCR for the amplification of DNA marker ladders in which the DNA marker ladders are amplified in a single PCR reaction instead of multi PCR reactions.

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

Under such circumstances, the present inventor has made intensive studies to develop a novel approach for preparing DNA size markers, in particular, enabling multiplex PCR to be practical for preparing DNA size markers and as a result have found a novel method for preparing DNA size markers in a cost and time effective manner by virtue of multiplex PCR. Accordingly, it is an object of this invention to provide a method for preparing DNA size markers by means of multiplex PCR.

It is another object of this invention to provide a method for preparing a plasmid set used in producing DNA size markers.

It is still another object of this invention to provide a DNA size marker set. It is further object of this invention to provide a plasmid set for producing a DNA size marker set.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjunction with the appended claims and drawings.

BREEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the 100 bp ladder products of this invention amplified by singlet polymerase chain reaction. Lanes 1-12 represent the ladders with indicated lengths of this invention and lane denoted 100 bp ladders represents 100 bp DNA ladders purchased from New England BioLabs, Inc.

Fig. 2 shows the 100 bp ladder products of this invention amplified by multiplex polymerase chain reaction. Lane 1 represents 100 bp DNA ladders purchased from New

England BioLabs, Inc. Lane 2 represents 100 bp ladders of this invention.

DETAILED DESCRDPTION OF THIS INVETNION

In one aspect of this invention, there is provided a method for preparing DNA size markers, which comprises the steps of: (a) selecting a plurality of specific regions on target DNA molecule(s) to be amplified so that each amplified product has a definite and desired length; (b) preparing a plurality of first primer pairs for amplifying the specific regions in which a 5 '-end portion of each forward primer of the first primer pairs has a common nucleotide sequence, a 5'- end portion of each reverse primer of the first primer pairs has a common nucleotide sequence and a 3 '-end portion of the first primer pairs has an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; (c) performing a polymerase chain reaction of the specific regions using the first primer pairs so that DNA molecules with definite and desired lengths are amplified; and (d) performing a multiplex polymerase chain reaction of the amplified products in the same reaction using one type of a third primer pair in which each third primer comprises a nucleotide sequence to be hybridized with a nucleotide sequence at each end portion of the amplified products.

The present invention is directed to a method for obtaining DNA size markers by multiplex polymerase chain amplification (hereinafter referred to as "PCR"), so that it allows providing DNA size markers with cost and time effectiveness. The success of this invention is ascribed mainly to employment of the first primer pair (forward and reverse primers) with a common nucleotide sequence at its 5 '-end portion. That is to say, a 5 '-end portion of each forward primer of the first primer pairs has a common nucleotide sequence and a 5 '-end portion of each reverse primer of the first primer pairs has a common nucleotide sequence. Therefore, the amplified products using the first primer pairs have the identical nucleotide sequences at their 5'- or 3'- end portions.

The first primer used in the present method comprises 5'- and 3 '-end portions with reference to a middle position of the primer. The term "5 '-end portion" used herein in conjunction with the first primer refers to a nucleotide sequence at the 5 '-end of the primer, preferably, having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the target DNA molecule(s). The term "3 '-end portion" used herein in conjunction with the first primer refers to a nucleotide sequence at the 3 '-end of the primer, having an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto.

Therefore, it is preferred that each of the first primer pair comprises: (i) the 3 '-end portion having an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; and (ii) the 5 '-end portion having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the target DNA molecule(s). The site on the specific region to anneal is located at a terminal portion of the specific region to be amplified. The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of the 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 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 the 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 3 '-end portion of the first primer has a nucleotide sequence substantially complementary to a site on a template DNA molecule. The term "substantially complementary" in reference to primer 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, such that the annealed primer can be extended by polymerase to form a complementary copy of the template. Therefore, it can be understood that this term has a different meaning from "perfectly complementary" or related terms thereof. It is appreciated that the 3 '-end portion can have one or more mismatches to a template to an extent that the ACP can serve as primer. Most preferably, the 3 '-end portion has a nucleotide sequence perfectly complementary to a site on a template, i.e., no mismatches.

According to a preferred embodiment, each of the first primer pair further comprises (iii) a regulator portion positioned between the 3 '-end portion and the 5 '-end portion comprising at least one universal base or non-discriminatory base analog, whereby the regulator portion is capable of enhancing an annealing specificity of the 3 '-end portion of the first primer to the site to anneal thereto.

Therefore, the preferred first primer has three portions: (i) the 3 '-end portion having an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; (ii) the 5 '-end portion having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the target DNA molecule(s); and (iii) a regulator portion positioned between the 3'-end portion and the 5'-end portion comprising at least one universal base or non-discriminatory base analog. The preferred first primer described above (called "annealing control primer") has been developed by the present inventor and disclosed in PCT/KR02/01781. The preferred first primer allows improving dramatically its annealing specificity to the site of interest, so that the amplified products having a definite and desired length can be obtained.

It is preferable that the regulator portion comprises contiguous nucleotides consisting of universal bases or non-discriminatory base analogs. The term "universal base or non- discriminatory base analog" used herein refers to one capable of forming base pairs with each of the natural DNA/RNA bases with little discrimination between them. The universal base or non- discriminatory base analog in the regulator portion includes 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'-OMe 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'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitro- benzimidazole, 2'-0-methoxyethyl 3 -nitropyrrole and combinations thereof, but not limited to.

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.

In a preferred embodiment, the first primer having a regulator portion contains at least 2 universal base or non-discriminatory base analog residues, more preferably, at least 3 universal bases or non-discriminatory base analogs. Advantageously, the universal base residues between the 3'- and 5 '-end portion sequences can be up to 15 residues in length. According to one embodiment, the first primer contains 2-15 universal base or non-discriminatory base analog residues. Most preferably, the universal bases between the 3'- and 5 '-end portion sequences are about 5 residues in length. The lengths of the 3'- and 5 '-end portion sequences of the first primer may vary. In a preferred embodiment, the 3 '-end portion is at least 6 nucleotides in length, which is considered a minimal requirement of length for primer annealing. More preferably, the 3 '-end portion sequence is from 10 to 25 nucleotides and can be up to 60 nucleotides in length.

In another preferred embodiment, the 5 '-end portion of the first primer contains at least 15 nucleotides in length, which is considered a minimal requirement of length for annealing under high stringent conditions. Preferably, the 5 '-end portion sequence can be up to 60 nucleotides in length. More preferably, the 5 '-end portion sequence is from 6 to 50 nucleotides, most preferably, from 20 to 25 nucleotides in length.

The 5 '-end portion has a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the template nucleic acid and this nucleotide sequence can serves as a priming site for subsequent amplification. The term "pre-selected arbitrary" nucleotide sequence used herein refers to any defined or pre-selected deoxyribonucleotide, ribonucleotide, or mixed deoxyribonucleotide sequence which contains a particular sequence of natural or modified nucleotides. In some embodiment, the pre-selected arbitrary nucleotide sequence of the 5 '-end portion can be composed of a universal primer sequence such as T3 promoter sequence, T7 promoter sequence, SP6 promoter sequence, and Ml 3 forward or reverse universal sequence. Using a longer arbitrary sequence (about 25 to 60 bases) at the 5 '-end portion reduces the efficiency of the first primer, but shorter sequences (about 15 to 17 bases) reduce the efficiency of annealing at high stringent conditions of the first primer. It is also a key feature of the present invention to use a pre-selected arbitrary nucleotide sequence at the 5 '-end portion of the first primer as a priming site for subsequent multiplex amplification. According to the present method, the nucleotide sequences of the 5 '-end portions of the forward primers of the first primer pairs may be identical to or different from those of the 5 '-end portions of the reverse primers.

In the present method, the target DNA molecule used may be derived from a wide variety of biological sources, including bacteriophages such as λ and 0X174, plasmids such as pBR322 and pUC18 and eucaryotic genomic DNAs obtained from yeast or mouse, the nucleotide sequences of which are known in the art. The specific regions to be amplified are present on different or same target DNA molecule(s). For example, where λ DNA is used as target DNA molecule, the specific regions are present on the same molecule whereas where genomic DNA from mouse is used, the specific regions are preferably present on different target DNA molecules.

The amplification of the specific regions is performed by PCR including cycles of denaturation, annealing and extension, more preferably, hot start PCR method known in the art. Methods known to denaturate double stranded DNA includes, but not limited to, heating, alkali, formamide, urea and glycoxal treatment, enzymatic methods (e.g., helicase action) and binding proteins. For instance, the denaturation can be achieved by heating at a temperature ranging from 80°C to 105°C. 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). Conditions of nucleic acid hybridization suitable for forming such double stranded DNA 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, URL Press, Washington, D.C. (1985).

A variety of DNA polymerases can be used in the amplification 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 such as obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus,

Thermococcus liter alis, and Pyrococcus furiosus (Pfu). Many of these polymerases may be isolated from bacterium itself or obtained commercially. Polymerase to be used with the subject invention can also be obtained from cells which express high levels of the cloned genes encoding the polymerase. 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 amplification reaction refers to an amount of each component such that the ability to achieve the desired amplification 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⁺, and dATP, dCTP, dGTP and dTTP in sufficient quantity to support the degree of amplification desired. All of the enzymes used in this amplification reaction may be active under the same reaction conditions. Indeed, buffers exist in which all enzymes are near their optimal reaction conditions. Therefore, the amplification process of the present invention can be done in a single reaction volume without any change of conditions such as addition of reactants. While the amplification of the specific regions can be performed according to conventional

PCR process, it can be also performed according to a two-stage PCR process for enhancing PCR specificity to target sequences, i.e., specific regions of interest. A two-stage PCR process has been suggested by the present inventor and disclosed in PCT/KR02/01781, accomplishing successfully the exclusion of non-specific PCR products.

Therefore, where the amplification is carried out according to a two-stage PCR process, the step (c) is followed by the step (c)' of a second-stage polymerase chain reaction using a second primer pair in which each second primer comprises a nucleotide sequence to be hybridized with a nucleotide sequence at each end portion of the amplified products in the step (c). In two-stage PCR process, the steps of (c) and (c)' are referred to as a first and second-stage PCR, respectively. The term "second primer" used herein refers to primer used in the second-stage PCR. According to a preferred embodiment, the second primer pair constitutes one primer having a substantially corresponding sequence to the 5 '-end portion of the forward primer of the first primer and the other primer having a substantially corresponding sequence to the 5 '-end portion of the reverse primer of the first primer. Thus, the first primer pair per se may be used as the second primer pair. More preferably, the second primer consists of a substantially corresponding sequence to the 5 '-end portion of the first primer.

The term "substantially corresponding" in reference to primer is used herein to mean that the primer comprises a completely or incompletely identical sequence to its compared sequence. Where the primer comprises an incompletely identical sequence, it may have one or more non- identical bases to an extent that it can serve as primer. For example, the second primer comprises completely or incompletely identical sequence to its compared sequence, i.e., the 5'- end portion of the first primer. Where the second primer comprises an incompletely identical sequence to the 5 '-end portion of the first primer, it may have one or more non-identical bases to an extent that it can serve as primer in the second-stage amplification.

It would be understood that if the nucleotide sequences of 5 '-end portions of the first primers are identical, one type of primer substantially corresponding to the sequence of 5 '-end portion will be used in the step of the second-stage amplification, whereas if they are different, two types of primer each substantially corresponding to the sequence of each 5 '-end portion of the first primer will be used in the step of the second-stage amplification.

Two amplification steps in the present methods are separated only in time. The first-stage amplification should be followed by the second-stage amplification. It would be understood that the first-stage amplification reaction mixture could include the second primers corresponding to the 5 '-end portion which will be used to anneal to the sequences of the 5 '-end portions of the first primer in the second-stage amplification, which means that the second primers corresponding to the 5 '-end portion can be added to the reaction mixture at the time of or after the first-stage amplification step.

As an alternative process, in the second-stage amplification step, the complete sequences of the first primer used in the first-stage amplification step, instead of the primers substantially corresponding to the 5 '-end portions of the first primer, can be used as primers under the high stringent conditions for re-amplifying the product generated from the first-stage amplification step, wherein the 3'- and 5'- ends of the product from the first amplification step which is generated from annealing and extension of the 3 '-end portion sequence of the first primer to the template nucleic acid under the low stringent conditions comprise the sequence or complementary sequence of the first primer and also serve as perfect paring sites to the first primer.

In this view, this alternative process is preferred because the process need not further add the primers substantially corresponding to the 5 '-end portions of the first primers to the reaction mixture at the time of or after the first-stage amplification step.

Annealing or hybridization in the amplification steps is performed under stringent conditions that allow for specific binding between a nucleotide sequence and primer. Such stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters. In the present methods, the second-stage amplification is generally performed under higher stringent conditions than the first-stage amplification. In a preferred embodiment, the first annealing temperature ranges from about 30°C to 68°C for the first-stage amplification step, more preferably, 40°C to 65 °C. It is preferred that the second annealing temperature ranges from about 50°C to 72°C for the second-stage amplification. According to a more preferred embodiment, the first annealing temperature is equal to or lower than the second annealing temperature.

According to the present methods, the first-stage amplification under low stringent conditions is carried out for at least 2 cycles of annealing, extending and denaturing to improve the specificity of primer annealing during the first-stage amplification, and through the subsequent cycles, the second-stage amplification is processed more effectively under high stringent conditions.

The first-stage amplification can be carried out up to 30 cycles. In a preferred embodiment, the first-stage amplification is carried out for 2 cycles. In another embodiment, the second-stage amplification under high stringent conditions is carried out for at least one cycle (preferably, at least 5 cycles) and up to 45 cycles to amplify the first-stage product. In a more preferred embodiment, the second-stage amplification is carried out for 25-35 cycles.

According to the present invention, the amplified products by singlet PCR described previously are re-amplified by multiplex PCR using a mixture of the amplified products and one type of a third primer pair. Advantageously, the amplified products in step (c) or (c)' are purified or isolated prior to multiplex PCR. This may be accomplished by gel electrophoresis, column chromatography, affinity chromatography or hybridization. In general, it is extremely difficult to set up PCR conditions to amplify more than 10 targets in parallel because an optimal PCR reaction is required to amplify even one specific locus without any unspecific by-products. Therefore, DNA size markers prepared by multiplex PCR have not been commercialized. However, the present invention provides DNA size markers by multiplex PCR. Successful multiplex PCR in this invention occurs due predominantly to the employment of one primer pair type that is ascribed originally to the common nucleotide sequence at 5 '-end portion of the first primer.

According to a preferred embodiment, the third primer pair used in multiplex PCR constitutes one primer having a substantially corresponding sequence to the 5 '-end portion of the forward primer of the first primer and the other primer having a substantially corresponding sequence to the 5 '-end portion of the reverse primer of the first primer. More preferably, the third primer consists of a substantially corresponding sequence to the 5 '-end portion of the first primer. It is the most preferred that the sequence of the third primer is perfectly corresponding, i.e., identical to the 5'-end portion of the first primer.- The nucleotide sequences of the third primers may be identical or different from each other.

The multiplex PCR in the present method may be performed in a similar manner to PCR described previously. It is preferred that the annealing temperature ranges from about 50°C to 72°C for the multiplex PCR.

Alternatively, the step (d) of multiplex polymerase chain reaction follows cloning into a plurality of plasmids the amplified products of the step (c) or (c)'. The cloning process may be carried out by a conventional method known in the art such as disclosed by Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001). The plasmid suitable in the cloning process includes a wide variety of cloning vectors commercially available such as pGEM-T Easy, pSClOl, ColEl, pBR322, pUC8/9, pHC79 and pUC19. The plasmids carrying the amplified products are transformed to suitable host cell such as E. coli and Bacillus sp. The transformed cells are then cultured in suitable media such as LB and the plasmids carrying insert sequences corresponding to DNA size markers of interest are obtained from the transformed cells. The multiplex PCR is carried out using a mixture of the cloned plasmids as template.

The product obtained from multiplex PCR is a mixture of DNA fragments covering base pair lengths of interest, giving DNA size markers ranging 20-1000 bp, 100-1500 bp, 200-6000 bp, 500-8000 bp, 1000-15000 bp, 2500-35000 bp, 50-10000 bp, etc. When the final products are visualized, a series of DNA segments can be found without a discontinuous marker and several gaps mainly corresponding to the smaller fragments. Preferably, the amplified products are of different and definite lengths ranging from 100 to

1,500 base pairs. More preferably, the final amplified products are visualized as a series of ladder with definite and desired lengths such as 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200 and 1500 bps. In addition, it is preferred that the final amplified products are of different and definite lengths ranging from 0.5 to 10.0 kilobase pairs. More preferably, the final amplified products are visualized as a series of ladder with definite and desired lengths such as

500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 8000 and 10000 bps.

In addition, the final amplified products can comprise at least one nucleotide with a label for detection. Suitable labels include, but not limited to, fiuorophores, chromophores, chemiluminescers, magnetic particles, radioisotopes, mass labels, electron dense particles, enzymes, cofactors, substrates for enzymes and haptens having specific binding partners, e.g., an antibody, streptavidin, biotin, digoxigenin and chelating group.

The present invention can provide a method for DNA size markers by means of multiplex PCR. According to the present method, the DNA size markers each of which has a definite and desired length are obtained with significant cost and time effectiveness. The DNA size markers prepared by the present method show neither discontinuous marker nor gaps corresponding to smaller fragments because the multiplex PCR in this method is performed under optimal conditions using one type of primer pair common to all the templates. In addition, the DNA fragments are prepared at nearly equal level by the present method, thus showing very similar band intensities on electrophoresis gel.

In another aspect of this invention, there is provided a method for preparing a plasmid set used in producing DNA size markers, which comprises the steps of: (a) selecting a plurality of specific regions on target DNA molecule(s) to be amplified so that each amplified product has a definite and desired length; (b) preparing a plurality of first primer pairs for amplifying the specific regions in which a 5 '-end portion of each forward primer of the first primer pairs has a common nucleotide sequence, a 5 '-end portion of each reverse primer of the first primer pairs has a common nucleotide sequence and a 3 '-end portion of the first primer pairs has an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; (c) performing polymerase chain reaction of the specific regions using the first primer pairs so that DNA molecules with definite and desired lengths are amplified; and (d) cloning into a plurality of plasmids the amplified products.

This method is aimed at providing a plasmid set used in producing DNA size markers. Each plasmid in the plasmid set carries an insert sequence corresponding to each DNA size marker with definite length.

Since the method of this invention is performed in the same manner as the method for preparing DNA size markers described above, except that the multiplex PCR is not carried out, the common descriptions between them are omitted in order to avoid the complexity of this specification leading to undue multiplicity.

This method can provide a plurality of cloned plasmids used in producing DNA size markers. The cloned plasmids are useful as a template in multiplex PCR. If the cloned plasmids are transformed to cells, the transformants can serve as a semi-permanent source for DNA size markers. Therefore, according to a preferred embodiment, the steps of transforming the cloned plasmids into host cells and culturing the transformed host cells are performed after the step (d).

In still another aspect of this invention, there is provided a DNA size marker set prepared by the method for preparing DNA size markers described above, in which the DNA size markers are of different and definite lengths and span the desired range of base pair lengths. In a further aspect of this invention, there is provided a plasmid set prepared by the method for preparing a plasmid set used in producing DNA size markers, in which the plasmids comprise insert sequences each having different and definite length and spanning the desired range of base pair lengths; in which when the insert sequences in the plasmids are amplified, a DNA size marker set having different and definite lengths and spanning the desired range of base pair lengths is produced.

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims.

EXAMPLES

In the experimental disclosure which follows, theses abbreviations apply: M (molar), mM

(millimolar), μM (micromolar), g (gram), μg (micrograms), ng (nanograms), 1 (liters), ml

(milliliters), μl (microliters), °C (degree Centigrade), Promega (Promega Co., Madison, USA),

QIAGEN (QIAGEN GmbH, Hilden, Germany), and Applied Biosystems (Foster City, CA, USA).

The oligonucleotide sequences used in the Examples are shown in Sequence Listing.

EXAMPLE 1: Genomic DNA Preparation

The liver tissues were excised from mouse (ICR) immediately, and then frozen quickly in liquid nitrogen. Two hundred micrograms of ground tissues were added to a 15 ml tube containing 1.2 ml of digestion buffer (100 mM NaCl, 10 mM Tris-Cl pH 8.0, 25 mM EDTA pH

8.0, and 0.5% SDS) and 6 μl of 20 mg/ml proteinase K. The tube was heated at 50°C for 12 hr.

The genomic DNA was extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1).

EXAMPLE 2: Cloning of Each 100 bp Ladder Fragment

In order to construct 100 bp DNA ladders, twelve different useful genes were used as templates for each fragment of the 100 bp ladder ranging in size from 100 bp to 1.5 kb, respectively. The mouse genomic DNA was used as a template for the amplification of each gene fragment.

A. Primer design A pair of annealing control primers (ACPs), which has been developed by the present inventor and disclosed in PCT/KR02/01781, for each gene was designed as follows:

1) 100 bp for Nitric oxide synthase (Nos-1) gene (Genbank Accession NO. L23806)

100-5 'primer: 5 ' -GTCTACCAGGCATTCGCTTCATimiGGACTGAGCTGTTAGAGAC-3 ' (SEQ ID NO: 1),

100-3 'primer: 5 '-CTGTGAATGCTGCGACTACGATimiGACCCAAGCGTGAGGAG-3 ' (SEQ ID NO:2);

2) 200 bp for CD4 antigen (cd4) gene (Genbank Accession NO. AF088189) 200-5 'primer: 5 '-GTCTACCAGGCATTCGCTTCATπiIICTCACGACCAGGCTTCC-3 ' (SEQ ID NO:3),

200-3 'primer: 5 ' -CTGTGAATGCTGCGACTACGATiπiIGCCAGTCCTTCCTTCAC-3 ' (SEQ ID NO:4);

3) 300 bp for p53 tumor suppressor gene (Genbank Accession NO. AF287146)

300-5 'primer: 5 '-GTCTACCAGGCATTCGCTTCATπiIICAGTCTACTTCCCGCCAT-3 ' (SEQ ID NO:5),

300-3'primer: 5'-CTGTGAATGCTGCGACTACGATmiIAGACCTGACAACTATCAACC-3' (SEQ ID NO:6);

4) 400 bp for Tumor necrosis factor (TNF) gene (Genbank Accession NO. M20155) 400-5 'primer: 5 '-GTCTACCAGGCATTCGCTTCATπ IICAACGGCATGGATCTCAA-3 '

(SEQ J_D NO:7),

400-3 'primer: 5 '-CTGTGAATGCTGCGACTACGATimiGACTCCAAAGTAGACCTGC-3 ' (SEQ ED NO:8),

5) 500 bp for Glyceraldehyde-3 -phosphate dehydrogenase (GAPD) gene (Genbank Accession NO. NM_008084)

500-5'primer: 5'-GTCTACCAGGCATTCGCTTCATimiCGTGGAGTCTACTGGTGTCT-3' (SEQ ID NO:9),

500-3 'primer: 5 '-CTGTGAATGCTGCGACTACGATmiICCACGACGGACACATTG-3 ' (SEQ ID NO: 10), 6) 600 bp for Plasmacytoma ABPC45 (12;15) translocated c-myc oncogene (Genbank Accession NO. K03229)

600-5 'primer: 5 ' -GTCTACCAGGCATTCGCTTCATiπiICCTCCTGCCTCCTGAAG-3 ' (SEQ ID NO: 11),

600-3 'primer: 5 '-CTGTGAATGCTGCGACTACGATimiAAAGAACACAGGGAAAGACC-3 ' (SEQ ID NO: 12);

7) 700 bp for ϊnterferon alpha 1 (MulFN- alpha 1) gene (Genbank Accession NO. X01974) 700-5'primer: 5'-GTCTACCAGGCATTCGCTTCATmiICTGTGCTTTCCTGATGGT-3'

(SEQ ID NO: 13),

700-3 'primer: 5 ' -CTGTGAATGCTGCGACTACGATIIIIITGAATGACAGTTTTGAGATG-3 ' (SEQ ID NO: 14);

8) 800 bp for MHC class 1 Q4 beta-2-microglobulin (Qb-1) gene (Genbank Accession NO.

M18837)

800-5 'primer: 5 ' -GTCTACCAGGCATTCGCTTCATππiCTCACTTTGTAGACCAGGC-3 ' (SEQ ID NO: 15),

800-3 'primer: 5 '-CTGTGAATGCTGCGACTACGATπiIIACAGACAGTAGCAATGTGG-3 '

(SEQ ID NO: 16);

9) 900 bp for Stem cell inhibitor/macrophage inflammatory protein la gene (Genbank Accession NO. X53372) 900-5 'primer: 5'-GTCTACCAGGCATTCGCTTCATmiIGTGTCCTACCCTGCTCAA-3' (SEQ

ID NO: 17),

900-3 'primer: 5 ' -CTGTGAATGCTGCGACTACGATIiπiTCTCTGGTATAAACAAAGCAT-3 ' (SEQ ID NO: 18);

10) 1000 bp for h terleukin-1 alpha gene (Genbank Accession NO. AF010237)

1000-5 'primer: 5'-GTCTACCAGGCATTCGCTTCATππiTTTAGGACATTCAGGTATCA-3' (SEQ ID NO: 19), 1000-3'primer: 5'-CTGTGAATGCTGCGACTACGATUπiTGAGGTAGGAAAGATGTAGC-3'

(SEQ ID NO:20);

11) 1200 bp for Granulocyte-macrophage colony stimulating factor gene (Genbank Accession NO. X03020)

1200-5 'primer: 5 '-GTCTACCAGGCATTCGCTTCATππiTAACATGTGTGCAGACCC-3 ' (SEQ ID NO:21),

1200-3 'primer: 5 ' -CTGTGAATGCTGCGACTACGATiππTGTCTTCCGCTGTCCAA-3 ' (SEQ ID NO:22);

12) 1500 bp for c-fos oncogene (Genbank Accession NO. V00727)

1500-5'primer: 5'-GTCTACCAGGCATTCGCTTCATIIIIITACTGACTGCACTTCCTGAC-3' (SEQ ID NO:23), and

1500-3 'primer: 5 '-CTGTGAATGCTGCGACTACGATIII1IACTAGGAACAACACACTCCA-3 ' (SEQ ID NO:24).

The Primers described previously were synthesized by means of a DNA synthesizer (Expedite 8900 Nucleic Acid Synthesis System, Applied Biosystems (ABI)) according to a standard protocol. The deoxyinosine in the primers was incorporated using deoxyinosine CE phosphoramidite (ABI). The primers were purified by means of an OPC cartridge (ABI), and their concentrations were determined by UV spectrophotometry at 260nm. In the primers described above, "I" symbolizes deoxyinosine.

The universal primers, JYC4 and JYC5, which correspond to the 5 '-end portion sequences of the 5' and 3' ACPs, respectively, are as follows: JYC4 5 '- GTCTACCAGGCATTCGCTTCAT -3 ' (SEQ ID NO:25), and

JYC5 5 '- CTGTGAATGCTGCGACTACGAT -3 ' (SEQ ID NO:26).

B. PCR amplification

Each fragment of the 100 bp ladder, ranging in size from 100 bp to 1.5 kb, was amplified by a two-stage PCR amplification using the above ACP set. The first-stage PCR amplification was performed by a hot start PCR method in which the procedure is to set up the complete reactions without the DNA polymerase and incubate the tubes in the thermal cycler to complete the initial denaturation step at >90°C (D'Aquila et al., Nucleic Acids Res., 19:3749 (1991)). Then, while holding the tubes at a temperature above 70°C, the appropriate amount of DNA polymerase can be pipetted into the reaction.

The first-stage PCR amplification was conducted by two cycles of PCR consisting of annealing, extending and denaturing reaction; the reaction mixture in a final volume of 49.5 μl containing 50 ng of the mouse genomic DNA, 5 μl of 10 x PCR reaction buffer (Promega), 5 μl of 25 mM MgCl₂, 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 1 μl of 5' ACP (10 μM) and 1 μl of 3' ACP (10 μM) was pre-heated at 94°C, while holding the tube containing the reaction mixture at the 94°C, 0.5 μl of Taq polymerase (5units/μl; Promega) was added into the reaction mixture; the PCR reactions comprise two cycles of 94°C for 40 sec, 55-60°C for 40 sec, and 72°C for 80 sec; followed by denaturing the amplification product at 94°C.

After the completion of the first-stage PCR amplification, each 1 μl of the universal primers, JYC4 (10 μM) and JYC5 (10 μM) which correspond to the 5 '-end portion sequences of the 5' and 3' ACPs, was added into the reaction mixture obtained from the first-stage PCR amplification, under denaturing temperature such as at 94°C. The second stage-PCR reaction was as follows: 35 cycles of 94°C for 40 sec, 68°C for 40 sec, and 72°C for 80 sec; followed by a 5 min final extension at 72°C.

C. Gel extraction and cloning The amplified products were analyzed by electrophoresis on a 2% agarose gel and detected by staining with ethidium bromide. The resulting PCR products can be also detected on a denaturing polyacrylamide gel by autoradiography or non-radioactive detection methods such as silver staining (Gottschlich et al., Res. Commun. Mol. Path. Pharm. 97:237-240(1997); and Kociok, N. et al., Mol. Biotechnol. 9, 25-33(1998)), the use of fluoresenscent-labelled oligonucleotides (Bauer et al. Nucleic Acids Res. 21 :4272-4280(1993); Ito, T., et al., FEBS Lett.

351:231-236(1994); Luehrsen, K.R. et al., BioTechniques 22:168-174(1997); and Smith, N.R. et al., BioTechniques 23:274-279(1997)), and the use of biotinylated primers (Korn et al., Hum. Mol. Genet. 1:235-242(1992); Tagle, D.A. et al., Nature 361:751-753(1993); and Rosok, O. et al., BioTechniques 21:114-121(1996)). After electrophoresis on agarose gel stained with EtBr, each PCR product was extracted using GENECLEAN U Kit (Q-BIOgene, USA) and cloned into the pGEM-T Easy vector (Promega, USA) as described by the manufacturer. The plasmid was transformed to the XLl-blue competent cell. The transformed cells were plated on LB/ampicillin agar plate. The plasmid was isolated from a single and white colony. The insert was confirmed by digestion with EcoRλ restriction enzyme. The plasmid with the insert was sequenced using ABI PRISM 310 genetic analyzer (Applied Biosystems, USA).

EXAMPLE 3: Amplification of 100 bp Ladders by Multiplex PCR

A. Mixture of each 100 bp clone

The isolated 100 bp ladder clones ranging in size from 100 bp to 1500 bp were mixed in a single tube by using 0.1-1 ng of each 100 bp ladder plasmid. The concentration of each 100 bp ladder plasmid was adjusted depending on the purpose; if the 500 bp and 1000 bp ladders are to be more amplified than others, more concentration of these two plasmids is added into the plasmid mixture. The mixture was diluted to 100-1000 fold with 1 X TE buffer. The diluted mixture was used as a template in the following multiplex PCR amplification.

B. Multiplex PCR

Multiplex PCR amplification was performed in 50 μl of reaction mixture containing 1 μl of the above diluted 100 bp ladder mixture, 5 μl of 10 x PCR reaction buffer (Promega), 5μl of 25 mM MgCl₂, 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 1 μl of 10 μM 5' universal primer (JYC4), 1 μl of 10 μM 3' universal primer (JYC5), and 0.5 μl of Taq polymerase (5 units/μl; Promega, USA). The amplification reaction was carried out in a

GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, USA) with denaturation for 3 min at 94°C, followed by 35 cycles of denaturation at 94°C for 40 sec, annealing at 68°C for 40 sec, and extension at 72°C for 40 sec, and a final extension at 72°C for 5 min. Two μl of the PCR reaction was detected on EtBr stained 2% agarose gel.

Figs. 1 and 2 show the amplified 100 bp ladder products by singlet PCR (Fig. 1) and multiplex PCR (Fig. 2), respectively. For the singlet PCR, each 100 bp ladder plasmid was used as a template for the amplification of the corresponding fragment by using the universal primers, JYC4 and JYC5, as described in the above multiplex PCR. The results indicate that the universal primers can each generate 100 bp ladder fragment from the corresponding 100 bp ladder plasmid. For the multiplex PCR, the mixture of each 100 bp ladder plasmid was used as templates for amplification of the multiplex 100 bp ladders as described above. The amplified 100 bp ladders were detected by staining with ethidium bromide (Fig. 2, lane 2). These results indicate that the method using ACPs for amplification of 100 bp ladder fragments ranging from 100 bp to 1500 bp by multiplex PCR approaches, produces desired molecular weight markers such as 100 bp ladders, without a discontinuous marker having several gaps mainly corresponding to the smaller fragment of the ladder, which was a major problem in the current multiplex PCR approaches for molecular weight markers (Amills et al., Genet. Anal., 13:147- 149 (1996)).

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 preparing DNA size markers, which comprises the steps of:

(a) selecting a plurality of specific regions on target DNA molecule(s) to be amplified so that each amplified product has a definite and desired length; (b) preparing a plurality of first primer pairs for amplifying the specific regions in which a 5 '-end portion of each forward primer of the first primer pairs has a common nucleotide sequence, a 5 '-end portion of each reverse primer of the first primer pairs has a common nucleotide sequence and a 3 '-end portion of the first primer pairs has an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto;

(c) performing polymerase chain reaction of the specific regions using the first primer pairs so that DNA molecules with definite and desired lengths are amplified; and

(d) performing multiplex polymerase chain reaction of the amplified products in the same reaction using one type of a third primer pair in which each third primer comprises a nucleotide sequence to be hybridized with a nucleotide sequence at each end portion of the amplified products.

2. The method according to claim 1, wherein the specific regions to be amplified are present on different or same target DNA molecule(s).

3. The method according to claim 1, wherein each of the first primer pair comprises: (i) the 3'- end portion having an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; and (ii) the 5 '-end portion having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the target DNA molecule(s).

4. The method according to claim 3, wherein each of the first primer pair further comprises (iii) a regulator portion positioned between the 3 '-end portion and the 5 '-end portion comprising at least one universal base or non-discriminatory base analog, whereby the regulator portion is capable of enhancing an annealing specificity of the 3 '-end portion of the first primer to the site to anneal thereto.

5. The method according to claim 4, wherein the regulator portion comprises contiguous nucleotides consisting of universal bases or non-discriminatory base analogs.

6. The method according to claim 4 or 5, wherein the universal base or non-discriminatory base analog is deoxyinosine, 1 -(2 '-deoxy-beta-D-ribofuranosyl)-3 -nitropyrrole or 5-nitroindole.

7. The method according to claim 4 or 5, wherein the regulator portion comprises at least two universal bases or non-discriminatory base analogs.

8. The method according to claim 1, wherein the nucleotide sequences of the 5 '-end portions of the forward primers of the first primer pairs are identical to or different from those of the 5'- end portions of the reverse primers.

9. The method according to claim 1, wherein the step (c) is followed by the step (c)' of a second-stage polymerase chain reaction using a second primer pair in which each second primer comprises a nucleotide sequence to be hybridized with a nucleotide sequence at each end portion of the amplified products in the step (c).

10. The method according to claim 9, wherein the second primer pair constitutes one primer having a substantially corresponding sequence to the 5 '-end portion of the forward primer of the first primer and the other primer having a substantially corresponding sequence to the 5 '-end portion of the reverse primer of the first primer.

11. The method according to claim 10, wherein the second primer consists of a substantially corresponding sequence to the 5 '-end portion of the first primer.

12. The method according to claim 9, wherein the step (c) is carried out by 2-4 cycles of polymerase chain reaction consisting of annealing, extension and denaturation.

13. The method according to claim 9 or 12, wherein the step (c)' is carried out by at least 5 cycles of polymerase chain reaction consisting of annealing, extension and denaturation.

14. The method according to claim 9, wherein the second-stage polymerase chain reaction is carried out with an annealing temperature of 50-72°C.

15. The method according to claim 1, wherein the multiplex polymerase chain reaction is carried out with an annealing temperature of 50-72°C.

16. The method according to claim 1, wherein the step (d) of multiplex polymerase chain reaction follows cloning into a plurality of plasmids the amplified products of the step (c) or (c)'.

17. The method according to claim 1, wherein the third primer pair constitutes one primer having a substantially corresponding sequence to the 5 '-end portion of the forward primer of the first primer and the other primer having a substantially corresponding sequence to the 5 '-end portion of the reverse primer of the first primer.

18. The method according to claim 17, wherein the third primer consists of a substantially corresponding sequence to the 5 '-end portion of the first primer.

19. The method according to claim 1 or 17, wherein the nucleotide sequences of the third primers are identical or different from each other.

20. The method according to claim 16, wherein the multiplex polymerase chain reaction is carried out using a mixture of the cloned plasmids as template.

21. The method according to claim 1, wherein the amplified products from multiplex polymerase chain reaction are of different and definite lengths ranging from 100 to 1,500 base pairs.

22. The method according to claim 1, wherein the amplified products from multiplex polymerase chain reaction are of different and definite lengths ranging from 0.5 to 10.0 kilobase pairs.

23. A method for preparing a plasmid set used in producing DNA size markers, which comprises the steps of: (a) selecting a plurality of specific regions on target DNA molecule(s) to be amplified so that each amplified product has a definite and desired length;

(b) preparing a plurality of first primer pairs for amplifying the specific regions in which a 5 '-end portion of each forward primer of the first primer pairs has a common nucleotide sequence, a 5 '-end portion of each reverse primer of the first primer pairs has a common nucleotide sequence and a 3 '-end portion of the first primer pairs has an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto;

(d) cloning into a plurality of plasmids the amplified products.

24. The method according to claim 23, wherein the specific regions to be amplified are present on different or same target DNA molecule(s).

25. The method according to claim 23, wherein each of the first primer pair comprises: (i) the 3 '-end portion having an annealing nucleotide sequence substantially complementary to a site on the specific region to anneal thereto; and (ii) the 5 '-end portion having a pre-selected arbitrary nucleotide sequence substantially not complementary to any site on the target DNA molecule(s).

26. The method according to claim 23, wherein each of the first primer pair further comprises (iii) a regulator portion positioned between the 3 '-end portion and the 5 '-end portion comprising at least one universal base or non-discriminatory base analog, whereby the regulator portion is capable of enhancing an annealing specificity of the 3 '-end portion of the first primer to the site to anneal thereto.

27. The method according to claim 26, wherein the regulator portion comprises contiguous nucleotides consisting of universal bases or non-discriminatory base analogs.

28. The method according to claim 26 or 27, wherein the universal base or non-discriminatory base analog is deoxyinosine, 1 -(2 '-deoxy-beta-D-ribofuranosyl)-3 -nitropyrrole or 5-nitroindole.

29. The method according to claim 26 or 27, wherein the regulator portion comprises at least two universal bases or non-discriminatory base analogs.

30. The method according to claim 23, wherein the nucleotide sequences of the 5 '-end portions of the forward primers of the first primer pairs are identical to or different from those of the 5 '-end portions of the reverse primers.

31. The method according to claim 23, wherein the step (c) is followed by the step (c)' of a second-stage polymerase chain reaction using a second primer pair in which each second primer comprises a nucleotide sequence to be hybridized with a nucleotide sequence at each end portion of the amplified products in the step (c).

32. The method according to claim 31, wherein the second primer pair constitutes one primer having a substantially corresponding sequence to the 5 ' -end portion of the forward primer of the first primer and the other primer having a substantially corresponding sequence to the 5 '-end portion of the reverse primer of the first primer.

33. The method according to claim 32, wherein the second primer consists of a substantially corresponding sequence to the 5 '-end portion of the first primer.

34. The method according to claim 31, wherein the step (c) is carried out by 2-4 cycles of polymerase chain reaction consisting of annealing, extension and denaturation.

35. The method according to claim 31 or 34, wherein the step (c)' is carried out by at least 5 cycles of polymerase chain reaction consisting of annealing, extension and denaturation.

36. The method according to claim 31, wherein the second-stage polymerase chain reaction is carried out with an annealing temperature of 50-72°C.

37. The method according to claim 23, wherein the cloned plasmids comprise insert sequences having different and definite lengths ranging from 100 to 1,500 base pairs.

38. The method according to claim 23, wherein the cloned plasmids comprise insert sequences having different and definite lengths ranging from 0.5 to 10.0 kilobase pairs.

39. A DNA size marker set prepared by the method of claim 1 , wherein the DNA size markers are of different and definite lengths and span the desired range of base pair lengths.

40. A plasmid set prepared by the method of claim 23 used in producing DNA size markers, wherein the plasmids comprise insert sequences being of different and definite lengths and spanning the desired range of base pair lengths; in which when the insert sequences in the plasmids are amplified, a DNA size marker set having different and definite lengths and span the desired range of base pair lengths is produced.