WO2005083121A1 - Procede d'amplification des elements d'une famille genique - Google Patents

Procede d'amplification des elements d'une famille genique Download PDF

Info

Publication number
WO2005083121A1
WO2005083121A1 PCT/KR2004/000426 KR2004000426W WO2005083121A1 WO 2005083121 A1 WO2005083121 A1 WO 2005083121A1 KR 2004000426 W KR2004000426 W KR 2004000426W WO 2005083121 A1 WO2005083121 A1 WO 2005083121A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
acp
sequence
gene family
site
Prior art date
Application number
PCT/KR2004/000426
Other languages
English (en)
Inventor
Jong-Yoon Chun
Original Assignee
Seegene, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seegene, Inc. filed Critical Seegene, Inc.
Priority to PCT/KR2004/000426 priority Critical patent/WO2005083121A1/fr
Priority to PCT/KR2004/002201 priority patent/WO2005083120A1/fr
Publication of WO2005083121A1 publication Critical patent/WO2005083121A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • 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.
  • 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.
  • degenerate primers mixture of primers
  • 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.
  • 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.
  • 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., M ⁇ ller, 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.
  • 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.
  • 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.
  • 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.
  • Fig. 2A shows a homeodomain consensus sequence generally recognized as the canonical 60 amid acid core by those sldlled 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.
  • 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. 3A 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).
  • HSD-ACP first homeobox specific degenerate annealing control primer
  • 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.
  • a method for amplifying members of a gene family which 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 the second primer used in step (c) and a third primer; in which said third primer comprises a hybridizing nucleot
  • 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).
  • GFSD-ACP novel gene family specific degenerate annealing control primer
  • 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.
  • ACP Annealing Control Primer
  • 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.
  • the first gene family specific degenerate annealing control primer 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 base 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.
  • 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.
  • 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.
  • 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.
  • primer 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.
  • 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.
  • dNMP naturally occurring dNMP
  • dAMP dAMP, dGMP, dCMP and dTMP
  • modified nucleotide or non-natural nucleotide.
  • 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.
  • 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.
  • 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.
  • 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-nitrobenzimid
  • 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 annealing temperature region due to its weaker hydrogen bonding interactions in base pairing.
  • 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.
  • 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.
  • 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.
  • the regulator portion of the first GFSD-ACP contains 2-10 universal base or non-discriminatory base analog residues.
  • 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-discriminatoiy base analog residues may be contiguous or intermittent, preferably, contiguous.
  • 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.
  • 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.
  • the site may comprise natural base(s) than universal bases for base paring specificity. For example, with referring to Fig.
  • 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.
  • the pre-selected nucleotide sequence of the 5 '-end portion is substantially not complementary to any site on the template nucleic acid.
  • the 5 '-end portion of the first GFSD-ACP contains at least 10 nucleotides in length.
  • 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.
  • 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 temperatures.
  • 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.
  • 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.
  • 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.
  • 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, streptavidin, biotin, digoxigenin and chelating group.
  • the 5 '-end portion also comprises bacteriophage 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 herein 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 amino 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.
  • 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.
  • 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.
  • the nucleotides "N” and “H” represent degeneracy (i.e., First-1(AI) represents a pool of 12 primers). It is preferred that the amplification for producing the primaiy 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.
  • nucleic acid as starting material is double-stranded
  • 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.
  • 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 Laboratoi ⁇ 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.
  • 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, HIV, 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, N.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.
  • 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 DNA polymerases can be used in the amplification step of the present methods, which includes "Klenow" fragment of E.
  • the polymerase is a thermostable DNA polymerase such as may be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Hiermis flavus, Thewnococcus literalis, and Pyrococcus furiosus (Pfu).
  • Taq Thermus aquaticus
  • Tth Thermus thermophilus
  • Thermus filiformis Hiermis flavus
  • Thewnococcus literalis Thewnococcus literalis
  • Pyrococcus furiosus Pyrococcus furiosus
  • the reaction mixture it is desirable to provide to the reaction mixture an amount of required cofactors such as Mg 2+ , 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 including all types of primers, the first GFSD-ACP and the third 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.
  • two amplification stages are carried out under different conditions, inter alia, at different annealing temperature each other.
  • the annealing in the first-stage amplification is performed under low stringent condition, inter alia, at low annealing temperature.
  • the first annealing temperature is between about 35°C and 55°C, still more preferably, 40-50°C and most preferably 45-50°C
  • the annealing portion of the first GFSD- ACP is restricted to the degenerate sequence, thereby improving annealing specificity.
  • 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.
  • 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 preferably performed under high stringent condition, inter alia, at high annealing temperature.
  • 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
  • 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 carried 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.
  • the second-stage amplification is carried out for 10-50 cycles, more preferably, 20-50 cycles, and most preferably, 20-40 cycles.
  • 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.
  • the first GFSD-ACP and the third primer are annealed to their complementary sequences on the template to generate the primary amplification product.
  • the regulator portion of the first GFSD-ACP comprises universal bases or non-discriminatory base analogs
  • its opposite strand on the primaiy amplification product comprises the nucleotides preferably recognized by DNA polymerase as described hereinafter.
  • 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 primary amplification product.
  • the nested amplification is carried out using the second GFSDN-ACP (fourth primer, nested primer) and the fifth primer.
  • the fourth primer i.e., the second GFSDN-ACP
  • 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 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 degener
  • the term "corresponding to" used herein with reference to two related nucleotide sequences is intended to express both perfectly and partially identical sequences to an extent that the one nucleotide sequence can be hybridized with a nucleotide sequence hybridizable 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.
  • 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 permissible, 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 formula II, 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., Tag polymerase) to direct the incorporation of natural dNMPs.
  • DNA polymerase e.g., Tag polymerase
  • 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.
  • 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.
  • 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 universal base or non-discriminatory base analog suitable in the regulator portion may include any base to show loss of discrimination when participating in DNA replication known in the art.
  • the universal base or non-discriminatory base analog suitable in the regulator portion of formula II could be described as the previous discussion for the regulator portion of formula I.
  • the regulator portion of the second GFSDN-ACP contains at least 3 universal bases or non-discriminatory base analog residues. According to one embodiment, the regulator portion of the second GFSDN-ACP contains 3-9 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 second GFSDN-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.
  • 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.
  • the nested amplification product is generated by the use of the second GFSDN-ACP overlapped significantly with the original primer, first GFSD-ACP. That is, the degenerate sequence portion 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 in 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 amino 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.
  • 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.
  • 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. 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 second GFSDN-ACP is at least 9 nucleotides in length coding for 3 amino acids.
  • 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.
  • the nucleotides "V” and "R” represent degeneracy (i.e., Second- l(WFQNE) represents a pool of 12 primers).
  • the nested amplification is preferably performed using the second GFSDN-ACP and the fifth primer under high stringent condition, inter alia, at high annealing temperature.
  • 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
  • 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 fifth primer work as a primer with higher specificity.
  • 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.
  • 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.
  • 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 fifth primer.
  • the nested amplification is carried out for 10-50 cycles, more preferably, 20-50 cycles, and most preferably, 20-40 cycles.
  • 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.
  • 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).
  • 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.
  • the step (a) for providing a sample of nucleic acids representing a population of mRNA transcripts can be conducted in accordance with the conventional procedures for the isolation of 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 3 '-end portion of the first primer comprises a hybridizing nucleotide sequence substantially complementary to a site of the mRNA to hybridize therewith.
  • the site of 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.
  • the 3'- end portion of the first primer comprises (i) a nucleotide sequence substantially complementary to a target known 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.
  • the first primer has a general fo ⁇ nula III: 5'-X" p .-Y" q -Z" r" -3' (in) 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 mRNA to hybridize therewith, p", q" and r" represent the number of nucleotides, and X", Y" and Z" are deoxyribonucleotide or ribonucleotide.
  • the pre-selected nucleotide sequence and the regulator portion can be described with adapting the descriptions of the first GFSD-ACP.
  • 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.
  • 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 franscriptase that has RNase H activity. If one uses an enzyme having RNase H activity, it may be possible to omit a separate RNase H digestion step, by carefully choosing the reaction conditions.
  • the oligo-dT primer has a general formula IV: 5'-X'V-Y'V-(dT) t -3' (IN) 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.
  • the oligo-dT primer of formula IN 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.
  • (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 third primer used in step (d) comprises a nucleotide sequence hybridizable 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).
  • the third primer is also represented by formula III.
  • a starting material is eucaryotic mRNAs containing poly A tail
  • the third primer is oligo-dT primer hybridizable to poly A tail.
  • the fifth primer used in step (e) could be described with adapting the discussion of the first primer.
  • the first, third and fifth primers could be substantially identical to or different from each other.
  • the first, third and fifth primers are substantially identical to each other in light of the easiness in their preparation.
  • the present method may be combined with many other processes known in the art to achieve a specific aim.
  • the isolation (or purification) of amplified product may follow the nested amplification. This can be accomplished by gel elecfrophoresis, column chromatography, affinity chromatography or hybridization.
  • the amplified product of this invention may be inserted into suitable vehicle for cloning.
  • 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 carries the amplified product under the control of, or operatively linked to a promoter.
  • the promoter used for procaryotic host includes, but not limited to, pL ⁇ 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.
  • 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.
  • 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.
  • recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, tobacco mosaic virus
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • 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 SH 2 domains) and G protein-coupled receptors (dopamine receptor), but not limited to.
  • the instant method is employed for detecting or analyzing a homeobox gene family.
  • gene families include envelope glycoprotein GP120; zinc finger, C2H2 type; leucine rich repeat; reverse franscriptase (RNA-dependent DNA polymerase) retroviral aspartyl protease; cytochrome b (N- terminal)/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 franscriptase thumb domain; and hepatitis C virus non-structural protein E2/NS1.
  • 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 conserveed 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 preferred embodiment, the present method is repeated using other first GFSD-ACP and second GFSDN-ACP for accommodating the occurring 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:l-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.
  • 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.
  • 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 (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'.
  • 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.
  • 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.
  • 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.
  • the use of degenerate primers in amplification makes it difficult to keep such a high annealing temperature.
  • 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.
  • 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.
  • 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.
  • the overlapping nested amplification in the present method greatly improves amplification specificity and sensitivity, resulting in the amplification of only desired products;
  • 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
  • Degenerate nested ACP system reduces degeneracy and the number of nested primers.
  • the degeneracy of the second GFSDN-ACP is reduced, which provides increased specificity of amplification. Furthermore, the number of the second GFSDN-ACP can be reduced, which saves a great amount of time and cost.
  • 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.
  • a primer for amplifying members of a gene family which is represented by the following general formula II: 5'-X' p .-S u -Y' v -Z' w -3 ' ( ⁇ ) 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 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 de
  • kits for amplifying members of a gene family which comprises the primer of formula I.
  • the subject kit further comprises the primer of formula II.
  • the kit of this invention further comprises the first primer, the third primer and/or the fifth 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.
  • the kits may also include various polynucleotide molecules, reverse franscriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • 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.
  • 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.
  • HSD-ACPs homeobox specific degenerate annealing control primers
  • HSD-ACPs 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.
  • 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.
  • a typical homeodomain has a consensus sequence of "NKIWF" at positions 45-49.
  • 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.
  • a typical homeodomain has a consensus sequence of "WFQ ⁇ R" at positions 48-52 and the degenerate sequence which encodes "WFQ ⁇ R" is designed for the 3 '-end portion of the second
  • 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 "Q ⁇ R” 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.
  • each HSD-ACPs is a pool of degenerate primers.
  • N (A, C, T, or G)
  • H (A, C, or T)
  • Y (C or T)
  • R (A or G)
  • I deoxyinosine
  • 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.
  • sequences of cDNA synthesis primer and oligo dT-ACP reverse primer are as follows:
  • 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.
  • Reverse transcription reaction was performed using the total RNAs for 1.5 hr at 42 °C in a reaction volume of 20 ⁇ l composed of the following: 3 ⁇ g of total RNA, 4 ⁇ l of 5 x reaction buffer (Promega, USA), 5 ⁇ l of dNTPs (each 2 mM), 2 ⁇ l of 10 ⁇ M cDNA synthesis annealing control primer (oligo (dT) ⁇ 8 -ACPl), 0.5 ⁇ l of RNase inhibitor (40 units/ ⁇ l, Promega), and 1 ⁇ l of reverse franscriptase (200 units/ ⁇ l, Promega) First-strand cDNAs were diluted by adding 80 ⁇ l of ultra-purified H 2 0.
  • 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 confrol 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 ⁇ l containing 50 ng of the first strand cDNA, 5 ⁇ l of 10 x PCR reaction buffer containing 15 mM MgCl 2 (Roche), 5 ⁇ l of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 3-8 ⁇ l of one of first HSD-ACPs (10 ⁇ M), 1 ⁇ l of oligo (dT) 15 -ACP2 (10 ⁇ M), and 0.5 ⁇ l of Taq polymerase (5units/ ⁇ l; 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
  • 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) ⁇ 5 -ACP2.
  • the PCR amplification was conducted in a final volume of 50 ⁇ l containing 3 ⁇ l of the first amplification products generated by the primary degenerate ACP- PCR, 5 ⁇ l of 10 x PCR reaction buffer containing 15 mM MgCl 2 (Roche), 5 ⁇ l of dNTP (2 mM each dATP, dCTP, dGTP, dTTP), 3-5 ⁇ l of one of second HSD-ACPs (10 ⁇ M), 1 ⁇ l of oligo (dT)i 5 -ACP2 (10 ⁇ M), and 0.5 ⁇ l of Taq polymerase (5units/ ⁇ l; 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.
  • 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) 18 -ACPl.
  • a primary ACP-PCR amplification was conducted using first HSD-ACPs, First-7(KI) (A) or First-12(QN) (B) , with oligo (dT) 15 -ACP2.
  • EXAMPLE 3 Isolation of Homeobox Member Genes Expressed Differentially Between Normal and Tumor Tissues of Human Brain and Stomach The present method was further applied to isolate members of the homeobox gene family expressed differentially between normal and tumor tissues of human 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 franscriptase, as described previously (Hwang, LT, 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.
  • ACP-PCR amplification for the identification of homeobox members expressed differentially between normal 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) ⁇ 8 -ACPl.
  • a primary degenerate ACP-PCR amplification was conducted using first HSD-ACP, First-9(KV) with (dT) 15 -ACP2.
  • a degenerate nested ACP-PCR amplification was conducted using second HSD-ACP, Second- 16- l(WFQNR), with (dT) I5 -ACP2.
  • Gtx a neuronal homeobox gene
  • Gtx a neuronal homeobox gene
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant d'amplifier les éléments d'une famille génique, plus précisément, un procédé permettant d'amplifier sélectivement des éléments d'une famille génique au moyen d'une nouvelle amplification nichée dégénérée avec un nouveau système d'amorce de commande de renaturation dégénérée spécifique aux gènes d'une famille, ainsi que ses applications.
PCT/KR2004/000426 2004-02-27 2004-02-27 Procede d'amplification des elements d'une famille genique WO2005083121A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/KR2004/000426 WO2005083121A1 (fr) 2004-02-27 2004-02-27 Procede d'amplification des elements d'une famille genique
PCT/KR2004/002201 WO2005083120A1 (fr) 2004-02-27 2004-09-01 Procede d'amplification de membres d'une famille de genes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2004/000426 WO2005083121A1 (fr) 2004-02-27 2004-02-27 Procede d'amplification des elements d'une famille genique

Publications (1)

Publication Number Publication Date
WO2005083121A1 true WO2005083121A1 (fr) 2005-09-09

Family

ID=34909905

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/KR2004/000426 WO2005083121A1 (fr) 2004-02-27 2004-02-27 Procede d'amplification des elements d'une famille genique
PCT/KR2004/002201 WO2005083120A1 (fr) 2004-02-27 2004-09-01 Procede d'amplification de membres d'une famille de genes

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/KR2004/002201 WO2005083120A1 (fr) 2004-02-27 2004-09-01 Procede d'amplification de membres d'une famille de genes

Country Status (1)

Country Link
WO (2) WO2005083121A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175749A1 (en) 2001-12-08 2003-09-18 Jong-Yoon Chun Annealing control primer and its uses
ES2400805T3 (es) 2006-05-04 2013-04-12 Seegene, Inc. Método para amplificar una secuencia de ADN desconocida adyacente a una secuencia conocida
KR20080037128A (ko) * 2006-10-25 2008-04-30 주식회사 씨젠 뉴클레오타이드 변이 검출 방법
WO2008143367A1 (fr) * 2007-05-21 2008-11-27 Seegene, Inc. Méthode d'haplotypage par amplification multiplex
US20120077716A1 (en) * 2010-09-29 2012-03-29 454 Life Sciences Corporation System and method for producing functionally distinct nucleic acid library ends through use of deoxyinosine
CN110819644A (zh) * 2019-11-27 2020-02-21 湖北工业大学 一种克隆法尼基转移酶基因家族成员群的方法
CN110878299A (zh) * 2019-11-27 2020-03-13 湖北工业大学 一种克隆异戊烯转移酶基因家族成员群的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001050A1 (fr) * 1987-07-31 1989-02-09 The Board Of Trustees Of The Leland Stanford Junior University Accroissement selectif de sequences de polynucleotides cibles
WO1993014217A1 (fr) * 1992-01-10 1993-07-22 Life Technologies, Inc. Utilisation de nucleotides predetermines possedant des caracteristiques modifies d'appariement de bases dans l'amplification de molecules d'acide nucleique
WO2003050305A1 (fr) * 2001-12-08 2003-06-19 Seegene, Inc. Amorce de commande de renaturation et ses utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001050A1 (fr) * 1987-07-31 1989-02-09 The Board Of Trustees Of The Leland Stanford Junior University Accroissement selectif de sequences de polynucleotides cibles
WO1993014217A1 (fr) * 1992-01-10 1993-07-22 Life Technologies, Inc. Utilisation de nucleotides predetermines possedant des caracteristiques modifies d'appariement de bases dans l'amplification de molecules d'acide nucleique
WO2003050305A1 (fr) * 2001-12-08 2003-06-19 Seegene, Inc. Amorce de commande de renaturation et ses utilisations

Also Published As

Publication number Publication date
WO2005083120A1 (fr) 2005-09-09

Similar Documents

Publication Publication Date Title
AU2002329104B2 (en) Annealing control primer and its uses
US10138518B2 (en) Annealing control primer and its uses
US5869251A (en) Use of primers containing nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules
CA2599647C (fr) Procedes faisant intervenir un oligonucleotide a double specificite et oligonucleotide a double specificite utilise
EP2365079A1 (fr) Procédés utilisant un oligonucléotide à double spécificité et oligonucléotide à double spécificité
JP4263612B2 (ja) アニーリング調節プライマーおよびその使用
JP2005511096A6 (ja) アニーリング調節プライマーおよびその使用
KR101231089B1 (ko) 기지의 서열에 인접한 미지의 ｄｎａ 서열을 증폭하는 방법
US8058032B2 (en) Method for amplifying unknown DNA sequence adjacent to known sequence
WO2005083121A1 (fr) Procede d'amplification des elements d'une famille genique
KR20030045124A (ko) 핵산 염기서열의 결정방법
WO2000020630A1 (fr) Amorces oligonucleotidiques destabilisant la formation de duplex non specifiques et leurs utilisations
WO2004063373A1 (fr) Marqueurs de taille d'adn et leur procede de preparation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 69(1) EPC, DATED 07-11-2006

122 Ep: pct application non-entry in european phase