WO2005083120A1 - Method for amplifying members of a gene family - Google Patents

Abstract

The present invention relates to a method for amplifying members of a gene family, in particular to a method for selectively amplifying members of a gene family by means of a novel degenerate nested amplification with a novel family gene-specific degenerate annealing control primer (ACP) system and its applications.

Description

METHOD FOR AMPLIFYING MEMBERS OF A GENE FAMILY

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for amplifying members of a gene family, in particular to a method for amplifying members of a gene family using novel family gene- specific degenerate annealing control primer and its applications.

DESCRIPTION OFTHE RELATEDART Gene families are groups of genes which are often functionally characterized by a particular type of function which the gene products in a cell undertake and which structurally have one or several conserved domains in common. Examples of gene families are the homeobox gene family as well as further transcription factor families. A common way to isolate members of a gene family from either cDNA or genomic DNA is to use PCR with degenerate primers (mixtures of primers) designed on the ^"basis of a conserved region (domain) sequence data from alignment of gene families. However, current available conventional methods for the isolation of members of a gene family using degenerate primers have inherent problems and limitations for the following reasons: (1) degenerate primer design is very difficult due to codon degeneracy and the additional degeneracy needed to represent multiple codons at a position in the alignment, (2) these degeneracies lead to complications in trying to find suitable annealing temperatures and degenerate primer length, (3) as the degeneracy increases to accommodate more divergent genes, the concentration of any single primer drops. As a result, the number of primer molecules in a PCR reaction that can prime synthesis during the amplification cycles drops, and these primers are used up early in the reaction, (4) artifactual amplification occurs because of primers in the pool which do not participate in amplification of the targeted gene but are available to prime non-specific synthesis, (5) the use of short primers required for short conserved regions needs low stringency annealing condition which exacerbate the above problems, and (6) the need to target regions of high sequence conservation containing codons of low degeneracy limits PCR amplification for members of gene family. Nested PCR most often leads to increased specificity as well as sensitivity of PCR amplification. It generally uses two primers that are internal to the product of the first PCR. The PCR product from the first PCR is used as template DNA for a second round of PCR with these internal primers. This should yield a smaller PCR product compared with the original product. Alternatively, extending one or both of the original primers by even two or three nucleotides at their 3 '-ends should be sufficient to impose increased specificity on the nested PCR. It is the 3'- end of the PCR primer that is most critical for determining specificity of PCR amplification. If the 3 '-nucleotide is not complementary to the template then no amplification should occur. So extending a nested PCR primer by two or three nucleotides should allow the specific target to be amplified but not the nonspecific product, even though the nested primers overlap significantly with the original primers (McPherson, M.J., Møller, S.G. (2000) Cloning genes by PCR, p. 213-240. In PCR. BIOS Scientific Publishers, Oxfordshire, UK). An obvious problem in nested PCR is the presence of the original template DNA. If the initial product is nonspecific and sufficient original template is present, nonspecific products may be generated during nested PCR. This problem cannot be avoided when nested PCR is applied for amplifying members of a gene family using degenerate primers. If the internal primers or nested primers anneal only to the first PCR product but not to the original template DNA during nested PCR, this problem will be overcome so that there should be no nonspecific amplification after the nested PCR. However, it is not possible with current conventional available nested PCR methods. Accordingly, the present inventor has studied to provide a method for amplifying members of a gene family in which the present invention method overcomes the problems and limitations of the state of the art such as the difficulty of degenerate primer design and PCR conditions, and amplification of nonspecific products, and in particular a method in which the nested PCR primers anneal only to the first PCR product but not to the original template DNA during nested PCR, eventually resulting in amplification of members of a gene family without nonspecific products. 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 To be free from the shortcomings of the conventional technologies described above, the present inventor has intensively researched to develop approaches capable of amplifying members of a gene family without false results by modifying annealing control primer (ACP) system, which has been developed by the present inventor and disclosed in WO 03/050305, and as a result found a novel method for amplifying members of a gene family by use of degenerate ACPs ingeniously designed, which permits to amplify members of a gene family in much more reliable and convenient manner. Accordingly, it is an object of this invention to provide a method for amplifying members of a gene family. It is another object of this invention to provide a degenerate primer for amplifying members of a gene family. It is still another object of this invention to provide a kit for amplifying members of a gene family. 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 Figs. 1A-1C schematically represent an embodiment of the present method showing a synthesis of first-strand cDNA by reverse transcription. A and B. First-stand cDNA is synthesized from mRNA having poly A tail such as eucaryotic mRNA. C. First-stand cDNA is synthesized from mRNA having no poly A tail such as procaryotic mRNA. Figs. ID schematically represent an embodiment of the present method showing a synthesis of a first-strand DNA from genomic DNA. Fig. 2A shows a homeodomain consensus sequence generally recognized as the canonical 60 amid acid core by those skilled in the art, which is described by Burglin, T.R. 1994 A comprehensive classification of homeobox genes. In: Duboule, D. (Ed.) Guidebook to the Homeobox Genes. Oxford University Press, New York, pp.27-71. Fig. 2B represents the structure of the degenerate annealing control primers used for amplifying members of a homeobox gene family in the present invention, which comprises degenerate sequences to be hybridized with the conserved region of homeodomains. Fig. 3 A schematically represents one specific embodiment of the present method showing a synthesis of first-strand cDNA by reverse transcription and a process for generating a primary amplification product using oligo dT-ACP and a first homeobox specific degenerate annealing control primer (HSD-ACP). Fig. 3B schematically represents one specific embodiment of the nested amplification process in the present method using the oligo dT-ACP and a second homeobox specific degenerate annealing control primer (HSD-ACP). Fig. 4 shows the amplified products generated by homeobox-specific ACP-PCR amplification for the isolation of homeobox members during mouse conceptus development. M denotes a molecular weight marker. The arrow heads are homeobox members amplified. Fig. 5 shows the amplified products generated by homeobox-specific ACP-PCR amplification for the identification of homeobox members expressed differentially between normal and tumor tissues of human brain and stomach. M denotes a molecular weight marker. The arrow heads are homeobox members amplified. Fig. 6 shows the amplified products generated by homeobox-specific degenerate nested ACP-PCR amplification for the identification of homeobox members using mouse genomic DNA as a template. M denotes a molecular weight marker. The arrow heads are homeobox members amplified.

DETAILED DESCRIPTION OF THIS INVETNION In one aspect of this invention, there is provided a method for amplifying members of a gene family of interest, which comprises the steps of: (a) providing a sample of nucleic acids; (b) synthesizing first strand DNA molecules from said nucleic acids using a first primer, in which the 3 '-end portion of said first primer comprises a hybridizing nucleotide sequence substantially complementary to a site of the nucleic acid to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur; (c) perfoπτiing a first-stage amplification of the first strand DNA molecules of members of the gene family of interest at a first annealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a second primer; wherein said second primer is a first gene family specific degenerate annealing control primer (first GFSD-ACP) which hybridizes with a first site of a conserved domain within the gene family of interest; in which said first annealing temperature enables said second primer to function as a primer, whereby second strand DNA molecules of members of the gene family are generated; (d) performing a second-stage amplification of the second strand DNA molecules of members of a gene family at a second annealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a third primer and a fourth primer; wherein said third primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the first primer sequence present at the 5 '-end of the first stand DNA molecules generated by step (b); in which said fourth primer comprises a nucleotide sequence corresponding to at least a partial sequence of said second primer, under conditions in which DNA amplification is achieved, resulting in a primary amplification product; and (e) selectively amplifying the primary amplification product by at least one cycle of primer annealing, primer extending and denaturing using a fifth primer and a sixth primer; in which said fifth primer is a second gene family specific degenerate nested annealing control primer (second GFSDN-ACP) which hybridizes with (i) a nucleotide sequence substantially complementary to at least a partial sequence of said first GFSD-ACP sequence present at the end of the primary amplification product and (ii) a second site of the conserved domain within the gene family of interest; wherein said second site includes an overlapped sequence with the first site of said conserved domain and a consecutive sequence of said conserved domain following the first site, under conditions in which DNA amplification is achieved, resulting in a nested amplification product; in which said sixth primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the third primer sequence present at the end of the primary amplification product generated by step (d).

The subject invention pertains to a unique method for amplifying members of a gene family using a novel gene family specific degenerate annealing control primer (hereinafter referred to as GFSD-ACP). The present method may be employed to amplify members of a gene family from any nucleic acid sample. Preferably, the method of this invention is used to identify members of a gene family of interest from gDNAs (genomic DNAs) or mRNAs. Where the present method is employed to amplify members of a gene family from mRNAs, the method comprises the steps of: (a') providing a sample of nucleic acids representing a population of mRNA transcripts; (b') synthesizing a population of first strand cDNAs from the mRNA transcripts using a first primer, in which the 3 '-end portion of said first primer comprises a hybridizing nucleotide sequence substantially complementary to a site of the mRNA to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur; (c') performing a first-stage amplification of the first strand cDNA molecules of members of the gene family at a first annealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a second primer; wherein said second primer is a first gene family specific degenerate annealing control primer (first GFSD-ACP) which hybridizes with a first site of a conserved domain within the gene family; in which said first annealing temperature enables said second primer to function as a primer, whereby second strand cDNA molecules of members of the gene family are generated; (d') performing a second-stage amplification of the second strand cDNA molecules of members of a gene family at a second annealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a third primer and a fourth primer; wherein said third primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the first primer sequence present at the 5'-end of the first strand cDNA generated by step (b'); in which said fourth primer comprises a nucleotide sequence corresponding to at least a partial sequence of said second primer, under conditions in which DNA amplification is achieved, resulting in a primary amplification product; and (e') selectively amplifying the primary amplification product by at least one cycle of primer annealing, primer extending and denaturing using a fifth primer and a sixth primer; in which said fifth primer is a second gene family specific degenerate nested annealing control primer (second GFSDN-ACP) which hybridizes with (i) a nucleotide sequence substantially complementary to at least a partial sequence of said first GFSD-ACP sequence present at the end of the primary amplification product and (ii) a second site of the conserved domain within the gene family; wherein said second site includes an overlapped sequence with the first site of said conserved domain and a consecutive sequence of said conserved domain following the first site, under conditions in which DNA amplification is achieved, resulting in a nested amplification product; in which said sixth primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the third primer sequence present at the end of the primary amplification product generated by step (d'). To overcome the shortcomings of the conventional approaches for amplifying members of a gene family, for example, the difficulty in designing degenerate primer and establishing optimal PCR conditions, and amplification of nonspecific products in nested PCR, the present inventor has modified the Annealing Control Primer (ACP) system, which has been developed by the present inventor and disclosed in WO 03/050305, to selectively amplify members of a gene family. All of the amplified products may represent members of a specific gene family in a sample to be analyzed. Since the ACP system is capable of dramatically improving amplification specificity, the use of ACP fundamentally prevents the non-specific priming of a primer during amplification and also simplifies the amplification process in this application. The features of the present invention lie in a unique degenerate nested ACP-PCR amplification procedure and primer design. According to a preferred embodiment, the first gene family specific degenerate annealing control primer (first GFSD-ACP) has a general formula I: 5'-X_p-Y_q- Z_r-3' (I) wherein, X_p represents a 5 '-end portion having a pre-selected nucleotide sequence, Y_q represents a regulator portion comprising at least two universal bases or non-discriminatory base analog residues, Z_r represents a degenerate sequence portion to hybridize with the first site of the conserved domain within the gene family, p, q, and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide. The first GFSD-ACP of this invention is developed for the amplification of a conserved domain within the gene family, using and modifying the principles of annealing control primers developed by the present inventor and disclosed in WO 03/050305, the teachings of which are incorporated herein by reference in its entity. The principle of the first GFSD-ACP is based on the composition of an oligonucleotide primer having 3'- and 5 '-end distinct portions separated by a regulator portion comprising at least two universal base or non-discriminatory base analog residues and the effect of the regulator portion on the 3'- and 5 '-end portions in the oligonucleotide primer. The presence of the regulator portion between the 3'- and 5 '-end portions of the first GFSD-ACP acts as a main factor, which is responsible for the improvement of primer annealing specificity. The term "nucleic acid" or "nucleotide" is a deoxyribonucleotide or ribonucleotide polymer in either single or double-stranded form, including known analogs of natural nucleotides unless otherwise indicated. The term "portion" used herein in conjunction with the primer of this invention refers to a nucleotide sequence separated by an intervening portion such as the regulator portion. The term "3 '-end portion" or "5 '-end portion" refers to a nucleotide sequence at the 3 '-end or 5 '-end of the primer of this invention, respectively, which is separated by the regulator portion. 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 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, dGMP, dCMP and dTMP), modified nucleotide or non-natural nucleotide. The term "substantially complementary" in reference to primer sequence is used herein to mean that the primer is sufficiently complementary to hybridize selectively to a nucleotide sequence under the designated annealing conditions, such that the annealed primer can be extended by polymerase to form a complementary copy of the nucleotide sequence. Therefore, this term has a different meaning from "perfectly complementary" or related terms thereof. It has been widely known that nucleotides at some ambiguous positions of degenerate primers have been replaced by universal base or a non-discriminatory analogue such as deoxyinosine (Ohtsuka et al, 1985, J. Biol. Chem. 260:2605-2608; Sakanari et al., 1989, Proc. Natl. Acad. Sci. 86:4863-4867), l-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (Nichols et al., 1994, Nature 369:492-493) and 5-nitroindole (Loakes and Brown, 1994, Nucleic Acids Res. 22:4039-4043) for solving the design problems associated with the degenerate primers because such universal bases are capable of non-specifically base pairing with all four conventional bases. However, there has not been any report that this universal base or a non-discriminatory analogue such as deoxyinosine, l-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and 5- nitroindole can be applied to a primer for amplifying members of a gene family as a regulator to discriminate each functional portion of a primer in accordance with annealing temperature. 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. According to a preferred embodiment, 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, inosine, 1 -(2' -deoxy-beta-D-ribofuranosyl)-3 -nitropyrrole or 5-nitroindole, most preferably, deoxyinosine. The presence of the regulator portion having universal bases such as deoxyinosines in a primer generates a low amiealing temperature region due to its weaker hydrogen bonding interactions in base pairing. As an extension of this theory, the present inventor has deduced that the presence of the regulator portion having universal bases between the 5 '-end portion and the degenerate sequence to hybridize with a conserved domain could generate a region which has a lower melting temperature, forms a boundary to each of the 5 '-end portion and the degenerate sequence portion of the primer, and facilitates the annealing of the degenerate sequence portion to the target conserved domain at specific temperature. This theory provides the basis of the first GFSD-ACP of this invention. The regulator portion in the first GFSD-ACP is capable of regulating an annealing portion (i.e., the degenerate sequence) of the primer in association with annealing temperature. This regulator portion prevents annealing of the 5 '-end portion sequence to a template and restricts the annealing portion of the primer to its degenerate sequence portion at the first annealing temperature. Consequently, the regulator portion dramatically improves annealing of the degenerate sequence portion of the first GFSD-ACP to the template. In a preferred embodiment, the regulator portion of the first GFSD-ACP contains at least 3 universal bases or non-discriminatory base analog residues between the 5 '-end portion and the degenerate sequence portion, more preferably, at least 4 universal bases or non-discriminatory base analogs. Advantageously, the universal base residues between the 5 '-end portion and the degenerate sequence portion of the first GFSD-ACP can be up to 10 residues in length. According to one embodiment, the regulator portion of the first GFSD-ACP contains 2-10 universal base or non-discriminatory base analog residues. Most preferably, the universal bases between the 5 '-end portion and the degenerate sequence portion of the first GFSD-ACP are about 3-6 residues in length. The presence of universal bases or non-discriminatory base analog residues may be contiguous or intermittent, preferably, contiguous. Alternatively, the regulator portion could be designed to serve as a supplementary annealing site to a partial sequence of the first site of the conserved domain or a sequence upstream of the first site of the conserved domain, together with exhibiting its inherent regulation function described above. Such dual function is advantageous if the regulator portion is at least 3, preferably at least 4, more preferably at least 5 and most preferably at least 6 universal base residues in length. Where the regulator portion is designed to have the supplementary annealing site, the site may comprise natural base(s) than universal bases for base paring specificity. For example, with referring to Fig. 2A and the sequences of the first HDS ACPs of Table 1 , the supplementary annealing site consists of "ITI" coding for valine or isoleucine at the position 45 of homeodomain consensus sequence described in Fig. 2A. The middle nucleotide "T" is incorporated for base paring specificity with the nucleotide sequence covering valine or isoleucine. The 5 '-end portion of the first GFSD-ACP contributes partially to improve the annealing specificity. Importantly, the 5 '-end portion serves alone or with other portions as a priming site in subsequent amplifications after the first-stage amplification. According to a preferred embodiment, the pre-selected nucleotide sequence of the 5 '-end portion is substantially not complementary to any site on the template nucleic acid. Generally, the 5 '-end portion of the first GFSD-ACP contains at least 10 nucleotides in length. 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 18 to 25 nucleotides in length. Using a longer sequence at the 5 '-end portion may reduce the efficiency of the first GFSD-ACP, but a shorter sequence may reduce the efficiency of annealing under high stringent conditions. As used herein the term "stringent condition" or "stringency" refers to the conditions of temperature, ionic strength (buffer concentration), and the presence of other compounds such as organic solvents, under which hybridization or annealing is conducted. As understood by those of skill in the art, the stringent conditions are sequence dependent and are different under different environmental parameters. Longer sequences hybridize or anneal specifically at higher tempeiatures. h some embodiment, the pre-selected nucleotide sequence of the 5 '-end portion of the first GFSD-ACP 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. According to one embodiment of the present invention, some modifications in the 5 '-end portion of the first GFSD-ACP can be made unless the modifications abolish the advantages of the first GFSD-ACP, i.e., improvement in annealing specificity. For example, the 5 '-end portion can comprises a sequence or sequences recognized by a restriction endonuclease(s), which makes it feasible to clone the amplified product into a suitable vector. In addition, the 5'- end portion can comprises at least one nucleotide with a label for detection or isolation of amplified product Suitable labels include, but not limited to, fluorophores, 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, streptavidm, biotm, digoxigenm and chelatmg group. The 5 '-end portion also comprises bacteπophage RNA polymerase promoter region. The degenerate sequence portion (i.e., 3 '-end portion) of the first GFSD-ACP is present at its 3 '-end, which hybridizes with the first site of the conserved domain. The term "conserved domain" used heiein refers to a segment of nucleotide sequence of a gene that is significantly similar between members of a gene family. The degree of similarity can vary. The term "degenerate sequence" refers to a pool of diverse nucleotide sequences encoding the identical one ammo acid sequence due to codon degeneracy. Thus, the degenerate sequence portion provides a pool of primers with various nucleotide sequences, at least one of which will be anticipated to anneal to the first site of a conserved domain. Although the degenerate sequence portion of the first GFSD-ACP hybridizes to the first site of the conserved domain, there are several partially complementary binding sites on the template. However, such nonspecific annealing can be considerably avoided by the unique structure of GFSD-ACP, inter alia, the effects of the regulator portion and 5 '-end portion. The length of the degenerate sequence portion may be determined based on various considerable factors, for example, the desirable amplification specificity and yield and the number of primer molecules covering the first site of the conserved domain. For example, if the degenerate sequence portion is designed to accommodate longer amino acid sequence, the degeneracy should increase to accommodate more divergent amino acid sequences. In this case, the amplification yield becomes lower due to dropped concentration of any single primer and the nonspecific amplification occurs at high frequency because of the dominance of primers in the pool which do not participate in annealing to the first site of the conserved domain. Generally, the 3 '-end portion of the first GFSD-ACP is at least 9 nucleotides in length coding for 3 amino acids. It is preferred that the annealing portion (i.e., the degenerate sequence) of the first GFSD-ACP is at least 12 nucleotides in length covering 4 amino acids, which may be considered the minimal requirement of length for primer annealing to amplify conserved domain with specificity. More preferably, the 3 '-end portion sequence is about from 9 to 30 nucleotides in length, still more preferably, about 11-21 nucleotides, and most preferably, about 11-15 nucleotides. The entire first GFSD-ACP is preferably from 25 to 80 nucleotides in length, more preferably, 30-60 nucleotides, and most preferably, 30-45 nucleotides. The first GFSD-ACP indicated herein refers to a pool of degenerate primers with various degenerate sequences due to codon degeneracy at its 3 '-end portion encoding the amino acid sequence of the first site of the conserved domain. The pool of degenerate primers is exemplified by SEQ ID NO:l of Table 1. In the nucleotide sequence of SEQ ED NO: 1, the nucleotides "N" and "H" represent degeneracy (i.e., First-l(AJ) represents a pool of 12 primers). It is preferred that the amplification for producing the primary amplification product is conducted by a two-stage amplification under different annealing temperature to maximize the advantages of the first GFSD-ACP system. The methods of the present invention can be used to amplify any desired nucleic acid molecule. Such molecules may be either DNA or RNA. The molecule may be in either a double-stranded or single-stranded form, preferably, double-stranded. 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, the strand separation can be achieved by heating at 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). The present method does not require that the molecules to be amplified have any particular sequence or length, hi particular, the molecules which may be amplified include any naturally occurring procaryotic, eucaryotic (for example, protozoans and parasites, fungi, yeast, higher plants, lower and higher animals, including mammals and humans) or viral (for example, Herpes viruses, HIN influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, etc.) or viroid nucleic acid. The nucleotide sequence can also be any nucleic acid molecule which has been or can be chemically synthesized. Thus, the nucleotide sequence may or may not be found in nature. The first GFSD-ACP used in the present invention is hybridized or annealed to the first site of the conserved domain so that a double-stranded structure is formed. Conditions of nucleic acid hybridization 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, Ν.Y.(2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). Hybridization of the first GFSD-ACP to the first site of the conserved domain is a prerequisite for its template- dependent polymerization with polymerases. Factors (see 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)) which affect the base pairing of the first GFSD-ACP to its complementary nucleic acids subsequently affect priming efficiency. The nucleotide composition of the first GFSD-ACP can affect the temperature at which annealing is optimal and therefore can affect its priming efficiency. A variety of DKSTA 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 thennostable DNA polymerase such as may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). When a polymerization reaction is 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 reaction conditions. Indeed, buffers exist in which all enzymes are near their optimal reaction conditions. It is preferred that two amplification stages of the present method are separated only in time. Therefore, the two-stage amplification can be conducted in a reaction using a combination of primer pairs, a pair of the first GFSD-ACP (second primer) and the third primer or a pair of the third and the fourth primer, or using all types of primers, the first GFSD-ACP (second primer), the third primer and the fourth primer. Annealing or hybridization in the two-stage amplification is performed under stringent conditions that allow for specific binding between a nucleotide sequence and the primers. Such stringent conditions for annealing will be sequence-dependent and varied depending on environmental parameters, hi the present methods, it is preferred that two amplification stages are carried out under different conditions, inter alia, at different annealing temperature each other. Preferably, the annealing in the first-stage amplification is performed under low stringent condition, inter alia, at low annealing temperature. More preferably, the first annealing temperature is between about 35°C and 55°C, still more preferably, 40-50°C and most preferably 45-50°C. At the first annealing temperature, the annealing portion of the first GFSD- ACP is restricted to the degenerate sequence, thereby improving annealing specificity. According to the present method, the first-stage amplification under low stringent conditions is carried out for at least one cycle 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. It is most preferred that the first-stage amplification is carried out for one cycle. One cycling of the first-stage amplification could fundamentally prevent the first GFSD- ACP alone from generating non-specific amplification products. During the one cycle of the first-stage amplification, the 3 '-end portion of the first GFSD-ACP binds to the first site of the conserved domain under such low stringent conditions. The second-stage amplification is carried out using a third primer and a fourth primer. The third primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the first primer sequence present at the 5 '-end of the first strand DNA molecules generated by step (b). Preferably, the third primer has a general formula IV: 5'-x"y-Y"v-z"v-3' (iv) wherein, X'"_p»> represents a 5 '-end portion having a pre-selected nucleotide sequence, Y'"_q-> represents a regulator portion comprising at least two universal bases or non- discriminatory base analog residues, Z'"_r- represents a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the first primer sequence present at the 5 '-end of the first strand DNA molecules generated by step (b), p'", q'" and r'" represent the number of nucleotides, and X'", Y'" and Z'" are deoxyribonucleotide or ribonucleotide. For the third primer of formula IV, the pre-selected nucleotide sequence and the regulator portion can be described with adapting the descriptions of the first GFSD-ACP. h a prefened embodiment, the 3 '-end portion of the third primer is at least 6 nucleotides in length, which is considered a minimal requirement of length for primer annealing. More preferably, the 3 '-end portion is from 10 to 25 nucleotides and can be up to 60 nucleotides in length. Most preferably, the nucleotide sequence of the third primer is substantially identical to that of the first primer in view of in light of conveniences in their preparation and execution of the present process. The fourth primer comprises a nucleotide sequence corresponding to at least a partial sequence of the second primer. The conesponding sequence to the second primer is required to have a suitable length which makes the fourth primer possible to serve as a primer under high stringent condition. Most preferably, the fourth primer is substantially identical to the second primer. In the most preferable embodiment, the second primer (first GFSD-ACP) in step (c) is identical to the fourth primer in step (d). The second-stage amplification is preferably performed under high stringent condition, inter alia, at high annealing temperature. Advantageously, the second annealing temperature is between about 50°C and 72°C, more preferably, about 55-70°C, and most preferably, about 60- 68°C. At a high annealing temperature such as the second annealing temperature, the 3 '-end portion of the first GFSD-ACP alone no longer serves as a priming site; instead, the all portions of the first GFSD-ACP and the third primer work as a primer with higher specificity. The second-stage amplification under high stringent conditions is earned out for at least one cycle, preferably, at least 5 cycles to generate the primary amplification product, using a combination of the first GFSD-ACP and the third primer, hi a more prefereed embodiment, the second-stage amplification is carried out for 10-50 cycles, more preferably, 20-50 cycles, and most preferably, 20-40 cycles. h the most preferable embodiment, the amplification is performed in accordance with PCR which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159. According to the second-stage amplification, the first GFSD-ACP and the third primer are annealed to their complementary sequences on the template to generate the primary amplification product. If the regulator portion of the first GFSD-ACP comprises universal bases or non-discriminatory base analogs, its opposite strand on the primary amplification product comprises the nucleotides preferably recognized by DNA polymerase as described hereinafter. For example, where the regulator portion of the first GFSD-ACP comprises at least two deoxyinosine or inosine residues, at least 2 deoxycytidine nucleotides are incorporated into its opposite strand on the pπmary amplification product. Following the generation of the primary amplification product, the nested amplification is carried out using the second GFSDN-ACP (fifth primer, nested primer) and the sixth primer. Accoidmg to a prefened embodiment, the fifth pnmer (i.e., the second GFSDN-ACP) has a general fonnula II: 5'-X'_p.-S_u-Y'_v-Z'_w-3' (II) wherein, X'_p> represents a 5 '-end portion having a nucleotide sequence corresponding to at least a partial sequence of the 5 '-end portion of the first GFSD-ACP, S_u represents a supplementary annealing portion comprising a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first GFSD-ACP in the amplified product of step (d),

Y'v lepresents a regulator portion comprising at least two universal bases or non-discπmmatory base analog residues for preventing annealing of X'_p and S_u portions to non-target sequences except to the nucleotide sequence complementary to the first GFSD-ACP, Z'_w represents a degenerate sequence portion to hybridize with a second site of the conserved domain within the gene family, the second site includes an overlapped sequence with the first site of said conserved domain and a consecutive sequence of the conserved domain following the first site, p', u, v and w represent the number of nucleotides, and X', S, Y', and Z' are deoxyribonucleotide or ribonucleotide.

The term "conespondmg to" used herein with reference to two related nucleotide sequences is intended to express both perfectly and partially identical sequences to an extent that one nucleotide sequence can be hybridized with a nucleotide sequence hybπdizable with the other comparative nucleotide sequence. The second GFSDN-ACP of the formula II has a nucleotide sequence corresponding to that of the first GFSD-ACP. The primer of the formula II exhibits much higher annealing specificity. The 5 '-end portion of the second GFSDN-ACP has a nucleotide sequence corresponding to the 5 '-end portion of the first GFSD-ACP. That is, the nucleotide sequence of the 5 '-end portion of the second GFSDN-ACP may be completely identical or partially identical to all or partial sequence of the 5 '-end of the first GFSD-ACP. Departures from complete identity are pennissible, so long as such departures are not sufficient to completely preclude hybridization to form a double-stranded structure between the 5 '-end portion of the second GFSDN-ACP and the nucleotide sequence complementary to the 5 '-end portion of the first GFSD-ACP. The supplementary annealing portion (S_u) is very unique in the second GFSDN-ACP of the fonnula π, which is partially responsible for the complete removal of the non-specific amplification. The supplementary annealing portion comprises a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first GFSD-ACP in the primary amplification product and thus provides the second GFSDN-ACP with stability in hybridization. This strategy for designing the supplementary annealing portion employs the recognition of universal base by DNA polymerase (e.g., Taq polymerase) to direct the incorporation of natural dNMPs. The recognition of universal base by DNA polymerase has been reported by Geoffrey C. Hoops, et al. (Nucleic Acids Research, 25(24):4866-4871(1997)), the teachings of which are incorporated herein by reference. For example, 8-hydroxyguanie, 2-hydroxyadenine, 6-O-methylguanine and xanthine direct the incorporation of (C and A), (T and A), (T and C) and (T and C), respectively. Furthermore, Geoffrey C. Hoops, et al. have resulted that a base having nitropyrrole and inosine direct most preferably the incorporation of dAMP and dCMP, respectively. Therefore, if the regulator portion of the first GFSD-ACP comprises at least two deoxyinosine or inosine residues, the supplementary annealing portion should comprise at least 2 deoxyguanosine nucleotides because deoxycytidine nucleotides are most preferably incorporated into the portion opposite to the regulator portion by Taq polymerase. In the second GFSDN-ACP, Y'_v, a regulator portion, prevents annealing of the X'_p. and S_u portions to non-target sequences except to the sequence complementary to the first GFSD-ACP. The regulator portion of formula II is hybridized with a part of the sequence opposite to the degenerate sequence portion of the first GFSD-ACP in the primary amplification product. Therefore, the regulator portion provides an additional annealing portion by way of the indiscriminative binding of universal bases or non-discriminatory base analogs. In this context, the regulator portion contributes to reduce the degeneracy so that the number of the second GFSDN-ACPs is decreased. The umveisal base or non-discrimmatory base analog suitable in the regulator portion may include any base to show loss of discrimination when participating in DNA replication known m the art. The universal base or non-discnmmatory base analog suitable in the regulator portion of fonnula II could be described as the previous discussion for the regulator portion of formula I h a prefened embodiment, the regulator portion of the second GFSDN-ACP contains at least 3 universal bases or non-discnmmatory base analog residues According to one embodiment, the regulator portion of the second GFSDN-ACP contains 3-9 universal base or non-discnmmatory base analog residues. Most preferably, the universal bases between the 5'- end portion and the degenerate sequence portion of the second GFSDN-ACP are about 3-6 residues m length The presence of universal bases or non-discnmmatory base analog residues may be contiguous or intermittent, preferably, contiguous. In the second GFSDN-ACP, Z'_w represents a degenerate sequence portion to hybridize with the second site of the conserved domain within the gene family and the second site includes an overlapped sequence with the first site of the conserved domain and a consecutive sequence of the conserved domain following the first site. As the final product of the piesent process, the nested amplification product is generated by the use of the second GFSDN-ACP overlapped significantly with the ongmal primer, first GFSD-ACP. That is, the degenerate sequence poition is annealed to the second site including an overlapped sequence with the first site of the conserved domain and a consecutive sequence of the conserved domain following the first site, so that the nested amplification product is generated m view of the conserved domain Such overlapping nested amplification is greatly responsible for the specific amplification of members of an interested gene family. Furthermore, this overlapping nested amplification covers a large range of ammo acid sequence of the conserved domain, which is sufficiently enough to isolate only target members of a gene family. The degenerate sequence portion of the second GFSDN-ACP is present at its 3 '-end, which hybridizes with the second site of the conserved domain. Thus, the second GFSDN-ACP described herein means a pool of primers with various nucleotide sequences, at least one of which will be anticipated to anneal to the second site of the conserved domain Although the degenerate sequence portion of the second GFSDN-ACP hybridizes to the second site of the conserved domain, there may be several partially complementary binding sites on the template. However, such nonspecific annealing can be significantly avoided by the influences of other portions in the second GFSDN-ACP. The length of the degenerate sequence portion may be determined based on various considerable factors, for example, the desirable amplification specificity and yield and the number of primer molecules covering the second site of the conserved domain. For example, if the degenerate sequence portion is designed to accommodate longer amino acid sequence, the degeneracy should increase to accommodate more divergent amino acid sequences, h this case, the amplification yield becomes lower due to dropped concentation of any single primer and the nonspecific amplification occurs at high frequency because of the dominance of primers in the pool which do not participate in annealing to the first site of the conserved domain. Generally, the 3 '-end portion of the second GFSDN-ACP is at least 9 nucleotides in length coding for 3 amino acids. It is preferred that the annealing portion (i.e., the degenerate sequence) of the second GFSDN-ACP is at least 12 nucleotides in length covering 4 amino acids, which may be considered the minimal requirement of length for primer annealing to amplify conserved domain with specificity. More preferably, the 3 '-end portion sequence is about from 9 to 30 nucleotides in length, still more preferably, about 11-21 nucleotides, and most preferably, about 11-15 nucleotides. It is preferred that the overlapped sequence with the first site of the conserved domain is the most conserved site. The length of the overlapped sequence is preferably at least 3 nucleotides, more preferably, 3-18 nucleotides, still more preferably, 3-15 nucleotides, and most preferably, 6-12 nucleotides. The entire second GFSDN-ACP is preferably from 25 to 80 nucleotides in length, more preferably, 30-60 nucleotides, and most preferably, 35-45 nucleotides. The second GFSDN-ACP indicated herein refers to a pool of degenerate primers with various degenerate sequences due to codon degeneracy at its 3 '-end portion encoding the amino acid sequence of the second site of the conserved domain. The pool of degenerate primers is exemplified by SEQ ID NO:21 of Table 2. In the nucleotide sequence of SEQ ID NO:21 the nucleotides "Y" and "R" represent degeneracy (i.e., Second- l(WFQNE) represents a pool of 12 primers). For the nested amplification, the sixth primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the third primer sequence present at the end of the primary amplification product generated by step (d). Preferably, the sixth primer has a general formula V: 5'-X""_p""-Y"' -Z""_r.—3 ' (V) wherein, X""_p">> represents a 5 '-end portion having a pre-selected nucleotide sequence, Y""_q-_" represents a regulator portion comprising at least two universal bases or non- discriminatory base analog residues, Z""_r"» represents a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the third primer sequence present at the end of the primary amplification product generated by step (d), p'", q'" and r'" represent the number of nucleotides, and X'", Y'" and Z'" are deoxyribonucleotide or ribonucleotide. For the sixth primer of formula V, the pre-selected nucleotide sequence and the regulator portion can be described with adapting the descriptions of the first GFSD-ACP. In a preferred embodiment, the 3 '-end portion of the sixth primer is at least 6 nucleotides in length, which is considered a minimal requirement of length for primer annealing. More preferably, the 3 '-end portion is from 10 to 25 nucleotides and can be up to 60 nucleotides in length. Most preferably, the nucleotide sequence of the sixth primer is substantially identical to that of the third primer in view of in light of conveniences in their preparation and execution of the present process. In the most preferable embodiment, the third primer in step (d) serves as the sixth primer with no further addition of primers. The nested amplification is preferably performed using the second GFSDN-ACP and the sixth primer under high stringent condition, inter alia, at high annealing temperature. Advantageously, the high annealing temperature is between about 55°C and 72°C, more preferably, about 60-70°C, and most preferably, about 65-68°C. At a high annealing temperature, the 3 '-end portion of the second GFSDN-ACP alone no longer serves as a priming site; instead, the all portions of the second GFSDN-ACP and the sixth primer work as a primer with higher specificity. Under such a high annealing temperature, the second GFSDN-ACP binds only to the target sites comprising the first GFSD-ACP sequence of the primary amplification products but not to any nonspecific sites of the primary amplification products or not to the original template DNA. Thus, the present invention method overcomes problems of nonspecific amplification resulting from the nonspecific binding of the nested primer, which is the major huddle of cunent conventional nested PCR. The nested amplification under high stringent conditions is carried out for at least one cycle, preferably, at least 5 cycles to generate the nested amplification product, using a combination of the second GFSDN-ACP and the sixth primer. In a more preferred embodiment, the nested amplification is carried out for 10-50 cycles, more preferably, 20-50 cycles, and most preferably, 20-40 cycles. The first primer in step (b), the third primer in step (d) and the sixth primer in step (e) could be substantially identical to or different from each other. Preferably, the first primer is identical to the third primer and/or the sixth primer. It is advantageous that the third primer is identical to the sixth primer. Most preferably, the first primer is substantially different from the third and sixth primers wherein the third and sixth primers are substantially identical to each other in light of conveniences in their preparation and execution of the present process. hi the most preferable embodiment, the nested amplification is performed in accordance with PCR which is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159. According to another embodiment of this invention, the present method further comprises the step of purifying the primary amplification product of step (d) to remove the second primer and third primer before performing step (e). For example, the purification of amplified product can be accomplished by gel electrophoresis, column chromatography, affinity chromatography or hybridization. It is most preferable that the purification be carried out using a spin column with silica-gel membrane. This method employs the selective binding properties of a silica-gel membrane to which the amplified products are adsorbed in the presence of high salt, while contaminants such as primer pass through the column. Therefore, the amplified products are quickly purified and obtained from the amplification reactions. In the present process, the step (a) for providing a sample of nucleic acids may be conducted in accordance with conventional procedures for the isolation of nucleic acid sample such as gDNA and mRNA (or total RNA) known to one of skill in the art (Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); Chomczynski, P. et al., Anal. Biochem., 167:156(1987); and Heikkila, J.J., Int. J. Biochem., 22: 1223(1990)). The first strand DNA molecules complementary to the nucleic acids are synthesized by use of the first primer and various template-dependent polymerases in accordance with conventional procedures. To synthesize a population of first strand cDNAs, a reverse transcription step using the first primer is conducted, details of which are found in Joseph Sambrook, et al., Molecular Cloning, A Laboratoiy Manual, Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, N.Y.(2001); and Noonan, K. F. et al., Nucleic Acids Res. 16:10366

(1988). The 3 '-end portion of the first primer comprises a hybridizing nucleotide sequence substantially complementary to a site of the nucleic acid (e.g., gDNA and mRNA) to hybridize therewith. The site of the nucleic acid (e.g., gDNA and mRNA) to hybridize includes, but not limited to, poly A tail, known sequence, unknown sequence, degenerate sequence deduced from amino acid sequence and conserved domain sequence of family gene. Therefore, the 3 '-end portion of the first primer comprises (i) a nucleotide sequence substantially complementary to a target lαiown sequence, (ii) an arbitrary sequence, (iii) a random sequence, (iv) a degenerate sequence deduced from amino acid sequence, (v) a nucleotide sequence substantially complementary to a conserved domain sequence of a gene family, or (vi) oligo-dT sequence. Preferably, the first primer has a general formula III: 5'-X"_p..-Y"_q..-Z"_r-3' (DOT) wherein, X"_p" represents a 5 '-end portion having a pre-selected nucleotide sequence, Y"_q" represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues, Z"_r" represents a hybridizing nucleotide sequence substantially complementary to a site of the nucleic acid to hybridize therewith, p", q" and r" represent the number of nucleotides, and X", Y" and Z" are deoxyribonucleotide or ribonucleotide. For the first primer of formula III, the pre-selected nucleotide sequence and the regulator portion can be described with adapting the descriptions of the first GFSD-ACP. hi a preferred embodiment, the 3 '-end portion of the first primer 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. Where the first primer is prepared to hybridize with poly A tail of mRNA, it is oligo-dT primer hybridizable to poly A tail. 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 a primer. Reverse transcription can be done with a 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. Most preferably, the oligo-dT primer has a general formula Dl': 5'-x'v-Y"_q..-( T)_r3' cmr) wherein, X"_p» represents a 5 '-end portion having a pre-selected nucleotide sequence, Y"_q" represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues, (dT)_t represents deoxythymidine nucleotides, p", q" and t represent the number of nucleotides, and X" and Y" are deoxyribonucleotide or ribonucleotide. For the oligo-dT primer of formula III', the pre-selected nucleotide sequence and the regulator portion can be described with adapting the descriptions of the first GFSD-ACP. The regulator portion highly enhances annealing specificity of (dT)_t to its target poly A tail. In a prefened embodiment, (dT)_t contains at least 6 dT nucleotides in length, which is considered a minimal requirement of length for primer annealing. More preferably, (dT)_t is from 10 to 20 dT nucleotides and can be up to 30 dT nucleotides in length. Most preferably, (dT) _t is about 15-20 dT nucleotides in length. The present method may be combined with many other processes known in the art to achieve a specific aim. For example, the isolation (or purification) of amplified product may follow the nested amplification. This can be accomplished by gel electrophoresis, column chromatography, affinity chromatography or hybridization. In addition, the amplified product of this invention may be inserted into suitable vehicle for cloning. Furthermore, the amplified product of this invention may be expressed in suitable host harboring expression vector. In order to express the amplified product, one would prepare an expression vector that canies the amplified product under the control of, or operatively linked to a promoter. Many standard techniques are available to construct expression vectors containing the amplified product and transcriptional/tanslational/control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. The promoter used for procaryotic host includes, but not limited to, pLλ promoter, trp promoter, lac promoter and T7 promoter. The promoter used for eucaryotic host includes, but not limited to, metallothionein promoter, adenovirus late promoter, vaccinia virus 7.5K promoter and the promoters derived from polyoma, adenovirus 2, simian virus 40 and cytomegalo virus. Certain examples of procaryotic hosts are E. coli, Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species, hi addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences. The expressed polypeptide from the amplified product may be generally purified with a variety of purposes in accordance with the method lαiown in the art. The present method is very effective to detect or analyze a gene family containing a conserved domain. The gene families to which the present method is applicable, include transcription factors, protein kinases (e.g., tyrosine kinases), phosphatases, ligands, receptors, proteases (e.g., metalloproteases), cytokines (e.g., interleukins), transmembrane proteins, adapter proteins (e.g., proteins containing SH₂ domains) and G protein-coupled receptors (dopamine receptor), but not limited to. Most preferably, the instant method is employed for detecting or analyzing a homeobox gene family. Particular examples of gene families include envelope glycoprotein GP120; zinc finger, C2H2 type; leucine rich repeat; reverse transcriptase (RNA-dependent DNA polymerase) retroviral aspartyl protease; cytochrome b (N- tenninal)/b6/petB; WD domain, G-beta repeat; ankyrin repeat; cytochrome C and quinol oxidase polypeptide I; immunoglobulin domain; NADH-ubiquinone/plastoquinone (complex I), various chains; cytochrome b(C-terminal)/b6/petD; ABC transporter; protein kinase domain; ribulose bisphosphate carboxylase large chain, catalytic domain; ribulose bisphosphate carboxylase large chain, N-terminal domain; TPR domain; PPR repeat; reverse transcriptase thumb domain; and hepatitis C virus non-structural protein E2 NS 1. The sequences and types of the gene families or conserved domains to which the present method is applied can be found at http://pfam.wustl.edu/browse.shtml or Conserved Domains Data Base of the National Center for Biotechnology Information. Accordingly, with reference to the information found in the accessible Data Base, the first GFSD-ACP and second GFSDN- ACP of this invention can be designed to have a degenerate sequence to be hybridized to the conserved domain of interest. According to a prefereed embodiment, the present method is repeated using other first GFSD-ACP and second GFSDN-ACP for accommodating the occuning frequency of the amino acid sequence in the conserved domain, so that most or all members of the gene family of interest are amplified. The combination of the pool of degenerate primers is exemplified by SEQ ID NOs: 1-20 for the first GFSD-ACP and SEQ ID NOs:21-43 for the second GFSDN- ACP in order to amplify most or all members of a homeobox gene family. For example, where the present method is applied to the selective amplification of homeodomain family, the first site of the conserved domain preferably spans the position 45- 50, more preferably, 46-49 of the amino acid sequence described in Fig. 2A. The second site of the conserved domain preferably spans the position 47-53, more preferably 48-52 of the amino acid sequence described in Fig. 2A. Most preferably, the first GFSD-ACP and second GFSDN- ACP for amplifying homeodomain family is represented by the structures depicted in Fig. 2B. That is, the first GFSD-ACP has the structure: 5'-(i) a pre-selected nucleotide sequence- (ii) a regulator portion comprising at least two universal bases-(iii) a degenerate sequence to hybridize with a coding sequence of K(Lys)I(Ile)W(Trp)F(Phe)-3 ' . The second GFSDN-ACP has the structure: 5'-(i) a pre-selected nucleotide sequence-(ii) a supplementary annealing portion comprising d(G)_n-(iii) a regulator portion comprising at least two universal bases-(iii) a degenerate sequence to hybridize with a coding sequence of W(Trp)F(Phe)Q(Gln)N(Asn)-3 ' . According to a specific embodiment, the first GFSD-ACP of SEQ ID NO: 7 and second GFSDN-ACP of SEQ ID NO:42 or 43 are the primer set with the highest probability to amplify a wide variety of members of homeodomain family. It would be recognized that the present invention takes a giant leap in the research field of a gene family in the senses that it provides perfectly true results as demonstrated in Examples hereinafter. The technologies showing no false results have not been suggested and even not been contemplated before the application of the present invention. In summary, the main benefits to be obtained from the use of the gene family specific degenerate (nested) annealing control primer system for amplifying members of a gene family are as follows: (a) Degenerate ACP system provides a high tolerance in primer search parameters. Degenerate primer design is usually very difficult because of codon degeneracy and the additional degeneracy needed to represent multiple codons at a position in the alignment. These degeneracies lead to complications in trying to find suitable annealing temperatures and primer lengths. However, the ACP system adapted in the present method allows the degenerate primer design for the 3 '-end degenerate portion highly tolerant in "primer search parameters" such as degeneracy, the length of degenerate portion, the melting temperature of the degenerate portion, and GC content; (b) Degenerate ACP system allows high annealing temperature. The first parameter to optimize the PCR conditions using degenerate primers is annealing temperature. Although it is important to keep the annealing temperature as high as possible to avoid extensive nonspecific amplification, the use of degenerate primers in amplification makes it difficult to keep such a high annealing temperature, hi contrast, the ACP system, utilizing a two-stage amplification, adapted in the present method allows the use of high annealing temperature, for example at least 10 to 20°C higher than the melting temperature of the degenerate portion. (c) Degenerate nested ACP-PCR improves specificity and sensitivity. The present invention method overcomes problems of nonspecific amplification resulting from the nonspecific binding of the nested primer, which is the major huddle of current conventional nested PCR. Under such a high annealing temperature, the second GFSDN-ACP binds only to the target sites comprising the first GFSD-ACP sequence of the primary amplification products but not to any nonspecific sites of the primary amplification products or not to the original template DNA. Overall, the overlapping nested amplification in the present method greatly improves amplification specificity and sensitivity, resulting in the amplification of only desired products; (d) Overlapping nested ACP-PCR generates a large range of amino acid sequence of the conserved domain, which is sufficiently enough to isolate only target members of a gene family; and (e) Degenerate nested ACP system reduces degeneracy and the number of nested primers. Since two codons for two amino acids in the 3 '-end degenerate portion of the first GFSD-ACP are replaced by six universal bases and used as a regulatory portion in the second GFSDN-ACP, the degeneracy of the second GFSDN-ACP is reduced, which provides increased specificity of amplification. Furthennore, the number of the second GFSDN-ACP can be reduced, which saves a great amount of time and cost. hi another aspect of this invention, there is provided a primer for amplifying members of a gene family, which is represented by the following general formula I: 5'-X_p-Y_q- Z_r-3' (I) wherein, X_p represents a 5 '-end portion having a pre-selected nucleotide sequence, Y_q represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues, Z_r represents a degenerate sequence portion to hybridize with a first site of a conserved domain within the gene family, p, q, and r represent the number of nucleotides, and

X, Y, and Z are deoxyribonucleotide or ribonucleotide. hi still another aspect of this invention, there is provided a primer for amplifying members of a gene family, which is represented by the following general formula IT: 5'-X'_p-S_u-Y'_v-Z'_w-3' (U) wherein, X'_p. represents a 5 '-end portion having a nucleotide sequence coreesponding to at least a partial sequence of the 5 '-end portion of the first GFSD-ACP, S_u represents a supplementary annealing portion comprising a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first GFSD-ACP in the amplified product of step (d), Y'v represents a regulator portion comprising at least two universal base or non-discriminatory base analog residues for preventing annealing of X'_p and S_u portions to non-target sequences except to the nucleotide sequence complementary to the first GFSD-ACP, Z'_w represents a degenerate sequence portion to hybridize with a second site of a conserved domain within the gene family, the second site includes an overlapped sequence with the first site of the conserved domain and a consecutive sequence of the conserved domain following the first site, p', u, v and w represent the number of nucleotides, and X', S, Y', and Z' are deoxyribonucleotide or ribonucleotide. Since the primers of this invention are employed in the present amplification process of members of a gene family described previously, the common descriptions between them are omitted in order to avoid the complexity of this specification leading to undue multiplicity.

In further aspect of this invention, there is provided a kit for amplifying members of a gene family, which comprises the primer of formula I. Preferably, the subject kit further comprises the primer of formula II. More preferably, the kit of this invention further comprises the first primer, the third primer and/or the sixth primer described hereinabove. The present kits may optionally include the reagents required for performing DNA amplification such as buffers, DNA polymerase, DNA polymerase cofactors, and deoxyribonucleotide-5 '-triphosphates. Optionally, the kits may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. Optimal amounts of reagents to be used in a given reaction can be readily determined by the skilled artisan having the benefit of the cunent disclosure. The kits, typically, are adapted to contain in separate packaging or compartments the constituents afore-described.

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, the following abbreviations apply: M (molar), mM (millimolar), μM (micromolar), g (grams), μg (micrograms), ng (nanograms), 1 (liter), ml (milliliters), μl (microliters), °C (degree Centigrade), Roche (Roche Diagnostics, Mannheim, Gennany), Promega (Promega Co., Madison, USA), Q-BIOgene(Qbiogene, Inc., Carlsbad, CA, USA), fnvitrogen (Carlsbad, USA), and Applied Biosystems (Foster City, CA, USA). The oligonucleotide sequences used in the Examples are shown in Sequence Listing and Tables 1 and 2.

EXAMPLE 1: Primer Design To demonstrate the application of a degenerate annealing control primer system for selectively amplifying members of a gene family, a homeobox gene family is used as a model in this invention, wherein the annealing control primers (ACPs) have been developed by the present inventor and disclosed in WO 03/050305. This is in no manner intended to limit the present invention to the specific gene families demonstrated. The multiple-aligned homeodomain sequences of available 4184 members of the homeobox gene family are obtained from existing on-line databases of homeobox website (http://pfam.wustl.edu/cgi- bin/getdesc?name=Homeobox). Among the homeodomain sequences of the 4184 members, only 3009 members showed full sequences of homeodomains but the others showed partial domain sequences. It was identified that the most conserved region is located on positions 45-52 within homeodomain by aligning amino acid sequences of homeodomains from a representative set (3009 full homeodomain sequences) of the homeobox gene family proteins. Thus, homeobox specific degenerate annealing control primers (HSD-ACPs) to be used for amplifying most or all members of the homeobox gene family are designed based on amino acid sequences located on the homeodomain positions 45-52. Two different HSD-ACPs were designed for degenerate nested ACP-PCR amplification in which first HSD-ACPs were used as an original primer for the primary PCR amplification and second HSD-ACPs, as a nested primer, was extended by additional nucleotides at the 3 '-end partially overlapping with the first HSD-ACPs. As a result, homeobox-specific members were selectively amplified during the nested PCR amplification. The 3 '-end target portion of the first HSD-ACPs has a degenerate sequence which is designed based on the amino acid sequences of homeodomain positions 45-49. For example, a typical homeodomain has a consensus sequence of "VK-WF" at positions 45-49. Most homeodomains have valine or isoleucine at position 45 and a ITI nucleotide (I = inosine) sequence covers all possible codons for valine (V) and isoleucine (I), GrTN and ATH (N = A,C,T,G,; H = A,C,T), respectively. Thus, the sequence at the 3 '-end portion of the first HSD- ACPs comprises the ITI plus the degenerate nucleotide sequence which encodes "KIWF". The 3 '-end target portion of the second HSD-ACPs has a degenerate sequence which is designed based on the amino acid sequences of homeodomain positions 48-52. For example, a typical homeodomain has a consensus sequence of "WFQNR" at positions 48-52 and the degenerate sequence which encodes "WFQNR" is designed for the 3 '-end portion of the second HSD-ACPs. Thus, the amino acid sequences at positions 48 and 49 such as "WF" represent the sequence overlapped between the first and second HSD-ACPs and the amino acid sequences at positions 50-52 such as "QNR" represent the sequence used for nested PCR amplification. The HSD-ACPs have a tripartite structure with a polydeoxyinosine linker between the 3'- end target binding sequence and the 5 '-end non-target sequence, wherein the 3 '-end target binding sequence comprises a degenerate sequence containing most or all of the possible nucleotide sequences encoding the conserved homeodomain positions as described above. Thus, each HSD-ACPs is a pool of degenerate primers.

The designed first HSD-ACPs sequences are found in Table 1. TABLE 1

Sequences of first Homeobox Specific Degenerate Annealing Control Primers (first HSD- ACPs)

SEQ

ID NO Designation Sequence Information I First-l(AI) 5 ' -GTCTACCAGGCATTCGCTTCATIIIITIGCNATHTGGTT-3 ' 2 First-2(AV) 5'-GTCTACCAGGCATTCGCTTCATIIIITIGCNGTNTGGTT-3'

3 First-3(EV) 5'-GTCTACCAGGCATTCGCTTCATIIIITIGARGTNTGGTT-3'

4 First-4(GN) 5'-GTCTACCAGGCATTCGCTTCATIIIITIGGNAAYTGGTT-3'

5 First-5(IT) 5'-GTCTACCAGGCATTCGCTTCATIIIITIATHACNTGGrT-3' 6 First-6(KF) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAARTTYTGGTT-3'

7 First-7(KI) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAARATHTGGTT-3'

8 Fιrst-8(KT) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAARACNTGGTT-3'

9 First-9(KV) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAARGTNTGGTT-3'

10 First-10(NN) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAAYAAYTGGTT-3' 11 First-ll(QI) 5'-GTCTACCAGGCATTCGCTTCATIIIITICARATHTGGTT-3'

12 First-12(QV) 5'-GTCTACCAGGCATTCGCTTCATIIIITICARGTNTGGTT-3'

13 First- -l(RV) 5'-GTCTACCAGGCATTCGCTTCATIIIITICGNGTNTGGTT-3'

14 Fιrst-13-2(RV) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAGNGTNTGGTT-3'

15 First-14-1(SN) 5'-GTCTACCAGGCATTCGCTTCATIIIITITCNAAYTGGTT-3' 16 First-14-2(SN) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAGNAAYTGGTT-3'

17 First- 15-1 (ST) 5 '-GTCTACCAGGCATTCGCTTCATIIIITITCNACNTGGTT-3 '

18 First-15-2(ST) 5'-GTCTACCAGGCATTCGCTTCATIIIITIAGNACNTGGTT-3'

19 First-16(TI) 5'-GTCTACCAGGCATTCGCTTCATIIIITIACNATHTGGTT-3'

20 First-17(W) 5'-GTCTACCAGGCATTCGCTTCATIIIITIGTNGTNTGGTT-3' N = (A, C, T, or G), H = (A, C, or T), Y = (C or T), R = (A or G), I = deoxyinosine

The designed second HSD-ACPs sequences are found in Table 2. TABLE 2

Sequences of second Homeobox Specific Degenerate Annealing Control Primers (second HSD- ACPs)

SEQ

ID NO Designation Sequence Information

21 Second-l(WFQNE)

22 Second-2(WFQNS) 5 ' '

23 Second-3(FFMNA) 5 '

24 Second-4( WFCNQ) 5 '

25 Second-5-l(WYQNR) 26 Second-5-2(WYQNR)

27 Second-6(WFGNK) 5 '

28 Second-7(WFQNA) 5 ' ' 29 Second-δ(WFANA) 5 '

30 Second-9(WFQNH) 5 '

31 Second-10(WFINQ) 5'-AGGCATTCGCTTCATGGGGTGπiIIITGGTTYATHAAYCA-3'

32 Second-ll(WFINA) 33 Second- 12- l(WFSNR)

34 Second-12-2(WFSNR) '

35 Second- 12-3 (WFSNR) 5 '

36 Second-12-4(WFSNR)

37 Second-13-l(WFCNR) 38 Second-13-l(WFCNR)

39 Second-14(WFQNK)

40 Second- 15-1 (WFKNR) 5 '

41 Second- 15-2(WFKNR) 5 ' '

42 Second-16-1 (WFQNR) 5 ' 43 Second-16-2(WFQNR)

N = (A, C, T, or G), H = (A, C, or T), Y = (C or T), R = (A or G), I = deoxyinosine

The first and second HSD-ACPs are used as forward primers and oligo dT-ACP primer is used as a reverse primer in homeobox-specific degenerate ACP-PCR amplification.

The sequences of cDNA synthesis primer and oligo dT-ACP reverse primer are as follows: (dT) , ₈-ACP 1 : 5 ' -CTGTGAATGCTGCGACTACGATIIIIITTTTTTTTTTTTTTTTTT-3 ' (SEQ ID NO:44) for cDNA synthesis; and (dT),₅-ACP2: 5'-CTGTGAATGCTGCGACTACGATIIIIITTTTTTTTTTTTTTT-3' (SEQ ID NO:45) for oligo dT-ACP.

EXAMPLE 2: Isolation of Homeobox Member Genes Expressed During Mouse Development The present invention was used to isolate members of the homeobox gene family involved in mouse development.

A. First-strand cDNA Synthesis Total RNAs from 4.5- and 18.5-dpc (E4.5 and El 8.5) conceptus tissues of mouse strain ICR were isolated and used for the synthesis of first-strand cDNAs by reverse transcriptase, as described previously (Hwang, I.T., et al., (2003) Annealing control primer system for improving specificity of PCR amplification. BioTechniques 35:1180-1184). Reverse transcription reaction was performed using the total RNAs for 1.5 hr at 42 ^°C in a reaction volume of 20 μl composed of the following: 3 μg of total RNA, 4 μl of 5 x reaction buffer (Promega, USA), 5 μl of dNTPs (each 2 mM), 2 μl of 10 μM cDNA synthesis annealing control primer (oligo (dT)_I8-ACPl), 0.5 μl of RNase inhibitor (40 units/μl, Promega), and 1 μl of reverse transcriptase (200 units/μl, Promega). First-strand cDNAs were diluted by adding 80 μl of ultra-purified H₂0.

B. Primary Degenerate ACP-PCR using first HSD-ACP Primary degenerate ACP-PCR was conducted into individual tubes, one each for each first HSD-ACP, by a two-stage PCR amplification to maximize the advantage of annealing control primer system, wherein the annealing control primer system has been developed by the present inventor and disclosed in WO 03/050305.

The two-stage PCR amplification was conducted at two different annealing temperatures in a final volume of 50 μl containing 50 ng of the first strand cDNA, 5 μl of 10 x PCR reaction buffer containing 15 mM MgCl₂ (Roche), 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 3-8 μl of one of first HSD-ACPs (10 μM), 1 μl of oligo (dT)₁₅-ACP2 (10 μM), and 0.5 μl of Taq polymerase (5units/μl; Roche); the tube containing the reaction mixture was placed in a preheated (94°C) thermal cycler; the first-stage PCR reaction consists of one cycle of 94°C for 5 min, 48°C for 3 min, and 72°C for 1 min, followed by the second stage-PCR reaction consisting of 30 cycles of 94°C for 40 sec, 65 °C for 40 sec, and 72°C for 40 sec, and followed by a 5 min final extension at 72°C.

C. Degenerate Nested ACP-PCR using second HSD-ACP To amplify only homeobox-specific members from the first amplification products, which might have non-specific products by the non-specific priming of the first HSD-ACP to the template since the degenerate sequence of the first HSD-ACP is too short to detect homeobox- specific members, a degenerate nested ACP-PCR was conducted using the second HSD-ACPs and the oligo (dT)ι₅-ACP2. The PCR amplification was conducted in a final volume of 50 μl containing 3 μl of the first amplification products generated by the primary degenerate ACP- PCR, 5 μl of 10 x PCR reaction buffer containing 15 mM MgCl₂ (Roche), 5 μl of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 3-5 μl of one of second HSD-ACPs (10 μM), 1 μl of oligo (dT)]₅-ACP2 (10 μM), and 0.5 μl of Taq polymerase (5units/μl; Roche); the tube containing the reaction mixture was placed in a preheated (94°C) thermal cycler; the PCR reactions consist of denaturing 94°C for 5 min, followed by 40 cycles of 94°C for 40 sec, 65 °C for 40 sec, and 72°C for 40 sec, and followed by a 5 min final extension at 72°C.

D. Gel Extraction 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. After electrophoresis on agarose gel stained with EtBr, each PCR product was extracted using

GENECLEAN II Kit (Q-BIOgene, USA). E. Cloning or Sequencing The extracted fragments were cloned into the pGEM-T Easy vector (Promega, USA), or TOPO TA Cloning Kit (Invitrogen, USA), as described by the manufacturer. The plasmids were transformed to the XL 1 -blue competent cell. The transformed cells were plated on LB/ampicillin agar plates. The plasmids were isolated from single and white colonies. The inserts were confirmed by digestion with EcoRX restriction enzyme. The plasmid with the insert was sequenced using ABI PRISM 310 genetic analyzer (Applied Biosystems, USA). Alternatively, the extracted fragments were used as templates for direct sequencing by the ABI PRISM 310 Genetic Analyzer. Fig. 4 shows the amplified products generated by homeobox-specific nested degenerate ACP-PCR amplification for the isolation of homeobox members during mouse conceptus development. The first-strand cDNAs are synthesized from total RNAs of mouse conceptus tissues at 4.5 (lane 1) and 18.5 (lane 2) [days postcoitus (dpc)] by using oligo (dT)_]8-ACPl. A primary ACP-PCR amplification was conducted using first HSD-ACPs, First-7(KI) (A) or First-12(QV) (B) , with oligo (dT)₁₅-ACP2. Using the primary ACP-PCR products as templates, a nested degenerate ACP-PCR amplification was conducted using second HSD-ACP, Second- 16-1 (WFQNR), with (dT)₁₅-ACP2. These products were turned out to be members of the homeobox gene family by sequence analysis. These results indicate that the homeobox-specific degenerate nested ACP-PCR method of the subject invention can be applied to amplify not only members of a homeobox family but also any other gene families. There were no false products. It means that all of the amplified products were members of homeobox genes. To my knowledge, so far there was no such a method only for selectively amplifying members of a gene family without the amplification of non-specific products. Furthermore, depending on first HSD-ACPs in the primary ACP-PCR amplification, members of different classes were amplified. For example, when First-7(KI) was used as a first HSD-ACP, most of the amplified products were Hox class members such as Hox A10, Hox CS , Box B7, Hox D10, Hox A7, and Hox A5, indicating that gene family-specific degenerate nested ACP-PCR system has an extremely high specificity enough to isolate only target class members of a gene family. The specificity of ACP-PCR system was already demonstrated in allele-specific PCR amplification by discriminating even a single-base mismatch for SNP genotyping using allele- specific ACPs in WO 03/050305 developed by the present inventor.

EXAMPLE 3: Isolation of Homeobox Member Genes E-xpressed Differentially Between Normal and Tumor Tissues of Human Brain and Stomach The present method was further applied to isolate memt ers of the homeobox gene family expressed differentially between normal and tumor tissues of Tiuman brain and stomach.

A. First-strand cDNA Synthesis Total RNAs from normal and tumor tissues of human brain and stomach were isolated and used for the synthesis of first-strand cDNAs by reverse transcriptase, as described previously (Hwang, IT., et al., (2003) Annealing control primer system for improving specificity of PCR amplification. BioTechniques 35: 1180-1184). First-strand cDNAs were synthesized as described in the First-strand cDNA Synthesis (Process A) of Example 2.

B. Primary Degenerate ACP-PCR using first HSD-ACP Primary degenerate ACP-PCR amplification was conducted as described in Example 2. C. Degenerate Nested ACP-PCR using second HSD-ACP To amplify only homeobox-specific members from the first amplification products, which might have non-specific products by the non-specific priming of the first HSD-ACP to the template since the degenerate sequence of the first HSD-ACP is too short to detect homeobox- specific members, a degenerate nested ACP-PCR was conducted using the second HSD-ACPs and the oligo (dT)ι₅-ACP2, as described in Example 2.

D. Gel Extraction The amplified products were analyzed as described in the Gel Extraction (Process D) of Example 2.

E. Cloning or Sequencing The extracted fragments were cloned as described in the Cloning or Sequencing (Process E) of Example 2. Fig. 5 shows the amplified products generated by homeobox-specific degenerate nested

ACP-PCR amplification for the identification of homeobox members expressed differentially between nonnal and tumor tissues of human brain and stomach. The first-strand cDNAs are synthesized from total RNAs of normal (lane 1) and tumor (lane 2) tissues of human brain (A) and stomach (B) by using (dT)₁₈-ACPl. A primary degenerate ACP-PCR amplification was conducted using first HSD-ACP, First-9(KV) with (dT)ι₅-ACP2. Using the primary ACP-PCR products as templates, a degenerate nested ACP-PCR amplification was conducted using second HSD-ACP, Second- 16-1 (WFQNR), with (dT)ι₅-ACP2. Total 15 fragments were amplified and all of them (arrow heads) were turned out to be members of homeobox gene family by sequence analysis. Among them, four members are novel brain tumor marker candidates. The rest of the members are lαiown homeobox genes in brain and stomach tissues. For instance, Gtx, a neuronal homeobox gene, has been reported to inhibit neuronal cell death (Hashimoto et al, 2004, J. Biol. Chem. (in press)). In addition, the loss of Gtx was observed in various malignant brain tumors (Lee et al, 2001, Mamm Genome. 12(2): 157-62). This observation is consistent with the results of the present invention in which the expression of Gtx was detected only in normal brain but not in tumor tissue.

EXAMPLE 4: Isolation of Homeobox Member Genes from genomic DNA The present method was further applied to isolate members of the homeobox gene family from mouse genomic DNA.

A. Synthesis of First-strand DNA Complementary to Genomic DNA Genomic DNA isolated from mouse placenta was used for the synthesis of first-strand DNAs by reverse transcriptase, as described in the First-strand cDNA Synthesis (Process A) of Example 2.

B. Primary Degenerate ACP-PCR using first HSD-ACP Primary degenerate ACP-PCR amplification was conducted as described in Example 2. C. Degenerate Nested ACP-PCR using second HSD-ACP Degenerate nested ACP-PCR amplification was conducted as described in Example 2.

D. Gel Extraction The amplified products were analyzed as described in the Gel Extraction (Process D) of Example 2.

E. Cloning or Sequencing The extracted fragments were cloned as described in the Cloning or Sequencing (Process E) of Example 2.

Fig. 6 shows the amplified products generated by homeobox-specific degenerate nested ACP-PCR amplification for the identification of homeobox members using mouse genomic DNA as a template. The first-strand DNAs are synthesized from mouse genomic DNAs by using (dT)ι₈-ACPl. A primary degenerate ACP-PCR amplification was conducted using first HSD-ACP, First-12(QV) with (dT)₁₅-ACP2. Using the primary ACP-PCR products as templates, a degenerate nested ACP-PCR amplification was conducted using second HSD-ACP, Second-16-l(WFQNR), with (dT)₁₅-ACP2. All of the amplified products were turned out to be members of homeobox gene family by sequence analysis.

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 amplifying members of a gene family of interest, which comprises the steps of: (a) providing a sample of nucleic acids; (b) synthesizing first strand DNA molecules from said nucleic acids using a first primer, in which the 3 '-end portion of said first primer comprises a hybridizing nucleotide sequence substantially complementary to a site of the nucleic acid to hybridize therewith, under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur; (c) performing a first-stage amplification of the first strand DNA molecules of members of the gene family of interest at a first amiealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a second primer; wherein said second primer is a first gene family specific degenerate annealing control primer (first GFSD-ACP) which hybridizes with a first site of a conserved domain within the gene family of interest; in which said first annealing temperature enables said second primer to function as a primer, whereby second strand DNA molecules of members of the gene family are generated; (d) performing a second-stage amplification of the second stand DNA molecules of members of a gene family at a second annealing temperature, comprising at least one cycle of primer annealing, primer extending and denaturing using a third primer and a fourth primer; wherein said third primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the first primer sequence present at the 5 '-end of the first strand DNA molecules generated by step (b); in which said fourth primer comprises a nucleotide sequence corcesponding to at least a partial sequence of said second primer, under conditions in which DNA amplification is achieved, resulting in a primary amplification product; and (e) selectively amplifying the primary amplification product by at least one cycle of primer annealing, primer extending and denaturing using a fifth primer and a sixth primer; in which said fifth primer is a second gene family specific degenerate nested annealing control primer (second GFSDN-ACP) which hybridizes with (i) a nxicleotide sequence substantially complementary to at least a partial sequence of said first GFSD-ACP sequence present at the end of the primary amplification product and (Ε) a second site of the conserved domain within the gene family of interest; wherein said second site includes an overlapped sequence with the first site of said conserved domain and a consecutive sequence of said conserved domain following the first site, under conditions in which DNA amplification is achieved, resulting in a nested amplification pro duct; in which said sixth primer comprises a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the third primer sequence present at the end of the primary amplification product generated by step (d).

2. The method according to claim 1, wherein said sample of nucleic acids represents a population of gDNAs (genomic DNAs) or mRNA transcripts.

3. The method according to claim 2, wherein said sample of nucleic acids represents a population of mRNA transcripts.

4. The method according to claim 1, wherein said first-stage amplifica-tion of step (c) is perfonned for one cycle.

5. The method according to claim 1, wherein said second-stage amplification of step (d) is perfonned for at least 5 cycles.

6. The method according to claim 1, wherein said first annealing temperature of step (c) is between about 35°C and 55°C.

7. The method according to claim 1, wherein said second annealing tempe-rature of step (d) is between about 50°C and 72°C.

8. The method according to claim 1, wherein said amplification of step (e) is performed at an annealing temperature of 50-72°C.

9. The method according to claim 1, wherein said fourth primer in step (d) is identical to the second primer in step (c).

10. The method according to claim 1, wherein said first GFSD-ACP has a general formula I: 5'-X_p-Y_q-Z_r-3' (I) wherein, X_p represents a 5 '-end portion having a pre-selected nucleotide sequence, Y_q represents a regulator portion comprising at least two universal bases or non-discriminatory base analog residues, Z_r represents a degenerate sequence portion to hybridize with the first site of the conserved domain within the gene family, p, q, and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide.

11. The method according to claim 1, wherein said second GFSDN-ACP has a general formula II: 5'-X'_p.-S_u-YVZ'_w-3' (II) wherein, X'_p. represents a 5'-end portion having a nucleotide sequence corresponding to at least a partial sequence of the 5 '-end portion of the first GFSD-ACP, S_u represents a supplementaiy annealing portion comprising a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first GFSD-ACP in the amplified product of step (d), Y'_v represents a regulator portion comprising at least two universal bases or non-discriminatory base analog residues for preventing annealing of X'_p and S_u portions to non-target sequences except to the nucleotide sequence complementary to the first GFSD-ACP, Z'_w represents a degenerate sequence portion to hybridize with a second site of the conserved domain within the gene family, the second site includes an overlapped sequence with the first site of said conserved domain and a consecutive sequence of the conserved domain following the first site, p', u, v and w represent the number of nucleotides, and X', S, Y', and Z' are deoxyribonucleotide or ribonucleotide.

12. The method according to claim 1, wherein said first primer has a general formula D3: 5'-X"_p"-Y"_q"-Z"_r-3' (Dl) wherein, X"_p" represents a 5 '-end portion having a pre-selected nucleotide sequence, Y"_q" represents a regulator portion comprising at least two universal bases or non-discriminatory base analog residues, Z"_r" represents a hybridizing nucleotide sequence substantially complementary to a site of the nucleic acid to hybridize therewith, p", q" and r" represent the number of nucleotides, and X", Y" and Z" are deoxyribonucleotide or ribonucleotide.

13. The method according to claim 1, wherein said third primer has a general formula IV: 5'-X'"_P".-Y'"_q...-Z'"_r.^,-3' (IV) wherein, X'"_p"> represents a 5'-end portion having a pre-selected nucleotide sequence, Y'"_q"> represents a regulator portion comprising at least two universal bases or non- discriminatory base analog residues, Z'"_r". represents a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the first primer sequence present at the 5 '-end of the first strand DNA molecules generated by step (b), p'", q'" and r'" represent the number of nucleotides, and X'", Y'" and Z'" are deoxyribonucleotide or ribonucleotide.

14. The method according to claim 1, wherein said sixth primer has a general formula V: 5'-X""_P".-Y""_q"..-Z""_r....-3' (V) wherein, X""_P"» represents a 5 '-end portion having a pre-selected nucleotide sequence, Y""_q"_" represents a regulator portion comprising at least two universal bases or non- discriminatory base analog residues, Z""_r"" represents a hybridizing nucleotide sequence to hybridize to a nucleotide sequence substantially complementary to the third primer sequence present at the end of the primary amplification product generated by step (d), p'", q'" and r'" represent the number of nucleotides, and X'", Y'" and Z'" are deoxyribonucleotide or ribonucleotide.

15. The method according to claim 12, wherein said first primer is identical to the third primer and/or the sixth primer.

16. The method according to claim 13, wherein said third primer is identical to the sixth primer.

17. The method according to claim 12, wherein Z'V- of said primer represented by formula III is oligo (dT) .

18. The method according to any one of claims 10-14, wherein said universal base or non- discriminatory base analog residue 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-nitropynole, 2'-F 3-nitropynOle, l-(2'-deoxy-beta-D- ribofuranosyl)-3-nitropynole, 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, mo holino-3 -nitropynole, phosphoramidate-5 -nitroindole, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4- nitrobenzimidazole, phosphoramidate-3 -nitropynole, 2'-0-methoxyethyl inosine, 2'0- methoxyethyl nebularine, 2'-0-methoxyethyl 5-nitroindole, 2'-0-methoxyethyl 4-nitrobenzimidazole, 2'-0-methoxyethyl 3-nitropynole, and combinations thereof.

19. The method according to 18, wherein said universal base or non-discriminatory base analog residue is deoxyinosine, inosine, 1 -(2 '-deoxy-beta-D-ribofuranosyl)-3 -nitropynole or 5- nitroindole.

20. The method according to 19, wherein said universal base or non-discriminatory base analog residue is deoxyinosine.

21. The method according to any one of claims 10-14, wherein said regulator portion comprise contiguous nucleotides having universal base or non-discriminatory base analog residue.

22. The method according to any one of claims 10-14, wherein p, p', p", p'" and p"" represent an integer of 10 to 60.

23. The method according to any one of claims 10-14 wherein q, u, v, q", q'" and q"" represent an integer of 2 to 10.

24. The method according to any one of claims 10-14, wherein r, w, r", r' ' ' and r" " represent an integer of 8 to 30.

25. The method according to claim 17, wherein t represents an integer of 10 to 30.

26. The method according to claim 20, wherein S comprises at least 2 contiguous deoxyguanosine nucleotides.

27. The method according to claim 1, wherein said gene family is selected from the group consisting of transcription factors, protein kinases, phosphatases, ligands, receptors, proteases, cytokines, transmembrane proteins, adapter proteins and G protein-coupled receptors.

28. The method according to claim 1, wherein said gene family is a homeobox gene family.

29. A primer for amplifying members of a gene family, which is represented by the following general fonnula I: 5'-X_p-Y_q- Z_r-3' (I) wherein, X_p represents a 5 '-end portion having a pre-selected nucleotide sequence, Y_q represents a regulator portion comprising at least two universal bases or non-discriminatory base analog residues, Z_r represents a degenerate sequence portion to hybridize with a first site of a conserved domain within the gene family, p, q, and r represent the number of nucleotides, and X, Y, and Z are deoxyribonucleotide or ribonucleotide.

30. A primer for amplifying members of a gene family, which is represented by the following general fonnula II: 5'-X'_p.-S_u-Y'_v-Z'_w-3' (II) wherein, X'_p. represents a 5'-end portion having a nucleotide sequence conesponding to at least a partial sequence of the 5 '-end portion of the first GFSD-ACP of claim 1, S_u represents a supplementaiy annealing portion comprising a nucleotide sequence to hybridize with a portion opposite to the regulator portion of the first GFSD-ACP in the amplified product of step (d) of claim 1, Y'_v represents a regulator portion comprising at least two universal bases or non- discriminatory base analog residues for preventing annealing of X'_p and S_u portions to non- target sequences except to the nucleotide sequence complementary to the first GFSD-ACP, Z'_w represents a degenerate sequence portion to hybridize with a second site of a conserved domain within the gene family, the second site includes an overlapped sequence with the first site of said conserved domain and a consecutive sequence of the conserved domain following the first site, p', u, v . and w represent the number of nucleotides, and X', S, Y', and Z' are deoxyribonucleotide or ribonucleotide.

31. A kit for amplifying members of a gene family, which comprises the primer of claim 29.

32. The kit according to claim 31 , wherein said kit further comprises the primer of claim 30.

33. The kit according to claim 31, wherein said kit further comprises the first primer, the third primer, and/or the sixth primer set forth in claim 1.

34. The kit according to claim 32, wherein said kit further comprises the first primer, the third primer and/or the sixth primer set forth in claim 1.