WO2018085584A1

WO2018085584A1 - A method of detecting nucleotide polymorphism associated with risk of developing psychosis and determining individual genotype

Info

Publication number: WO2018085584A1
Application number: PCT/US2017/059784
Authority: WO
Inventors: Perry CHAN
Original assignee: Vitalq, Inc.
Priority date: 2016-11-02
Filing date: 2017-11-02
Publication date: 2018-05-11

Abstract

A method for conducting an allele- specific real time polymerase chain reaction to detect a nucleotide polymorphism, NC_000014.8:g.105239192T>C, of the v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1) gene including the steps: a. obtaining a genomic DNA sample from a test subject; b. subjecting said genomic DNA sample to two separate polymerase chain reactions; c.utilizing in said one of the polymerase chain reactions with a primer specific to direct amplification of the T-allele with the following sequence: SEQ1: T- Allele 5 ' -GGGGATGGAGGTGTAGCCCGT-3 '; d. utilizing in said the other polymerase chain reactions with a primer specific to direct amplification of the C-allele with the following sequence: SEQ2: C-Allele: 5 ' -GGGGATGGAGGTGTAGCCCGC-3 '; e. utilizing in said both polymerase chain reactions with a primer to direct amplification of either alleles with the following sequence: SEQ3: 5'- GCGGTCGCCTGCCCTTCTACAACC-3'; and f. utilizing a composition for amplifying and monitoring a target nucleic acid sequence in the sample.

Description

SPECIFICATION

TITLE

A METHOD OF DETECTING NUCLEOTIDE POLYMORPHISM ASSOCIATED WITH RISK OF DEVELOPING PSYCHOSIS AND DETERMINING INDIVIDUAL

GENOTYPE

FIELD OF THE INVENTION

The present disclosure relates to a method of detecting nucleotide polymorphism associated with risk of developing cannabis-induced psychosis and determining individual genotype. BACKGROUND OF THE INVENTION

The use of cannabis is associated with an increased risk of schizophrenia. However, only a small minority of individuals who use the drug will develop psychotic symptoms. Since behavior change, e.g., abstaining from cannabis use can be effective in minimizing the risk of developing psychotic symptoms, it is imperative to develop a means to identify those at-risk individuals. While environmental factors can contribute to the risk, genetic susceptibility to developing psychosis symptoms in association with the use of cannabis is an important underlying factor. Supportive evidence to genetic susceptibility include: increased sensitivity to the psychotomimetic effects of cannabis in individuals with a family history of schizophrenia; and 15 times greater positive schizotypal symptoms from recent cannabis use in unaffected siblings of patients with schizophrenia compared with controls.

Recently a new candidate gene, AKTl, that is associated with such genetic susceptibility to cannabis-induced psychosis, has been identified. Various names have been given to the AKTl gene, including: RAC-alpha serine/threonine -protein kinase, v-Akt murine thymoma viral oncogene homolog 1, v-Akt murine thymoma viral oncogene-like protein 1, protein kinase B alpha, proto-oncogene c-Akt, RAC-PK-alpha, PKB alpha, protein kinase B, PKB -ALPHA, AKT, AKTlm, PKB, RAC, CWS6, PRKBA and RAC- ALPHA. Herein, the nomenclature AKTl will be used. AKTl is a promising target as delta-9-tetrahydrocannbinol (Delta-9-THC), has been shown to activate AKTl in vitro and in vivo. THC is the primary psychoactive ingredient in marijuana and depending on the particular plant, either THC or cannabidiol (CBD) is the most abundant cannabinoid in marijuana. Increasingly, however, at least for marijuana being cultivated for recreational use, higher ratios of Delta-9-THC are targeted. Delta-9-THC and also Delta-8-THC are the only compounds in the marijuana plant that produce all the psychoactive effects of marijuana. Because Delta-9-THC is much more abundant than Delta-8-THC, the psychoactivity of marijuana has been attributed largely to the effects of Delta-9-THC. Secondly, AKT1 is located primarily in the hippocampus, striatum and cerebellum and the AKT1 kinase protein forms part of the striatal dopamine receptor signaling cascade and hence has a plausible biological mechanism for interacting with cannabis to confer an increased risk of schizophrenia.

Several studies have recently been published to further substantiate the association. In one study with 801 patients with schizophrenia and 740 unaffected siblings, the AKT1 rs2494732 polymorphism (c.l 172+23 A>G or NC_000014.8:g.l05239192T>C) (FIG. 1), was among the 152 genetic markers studied that was shown to associate with psychosis development and cannabis use. Carriers of two copies of the C allele of rs2494732 (single- nucleotide polymorphism) of the AKT1 gene had a two-fold greater risk of being diagnosed with a psychotic disorder or having greater schizotypy siblings if they had used cannabis compared to carrier with two copies of the T allele. In another study of 489 patients with first-episode psychosis, carriers of the C/C genotype with a history of cannabis use were again shown to have greater than two-fold risk of developing a psychotic disorder compared to carriers of the T/T genotype. Cognitive impacts of this polymorphism have also been observed with cannabis-using psychotic patients who are carriers of the AKT1 rs2494732 C/C genotype performing more poorly on a task of sustained attention than T/T carriers. In another study of a total 442 healthy young cannabis users while intoxicated with their own cannabis, variation at the rs2494732 locus of the AKT1 gene predicted acute psychotic response to cannabis along with dependence on the drug and baseline schizotypal symptoms.

Popular methods to determine the genotype of individuals, such as single base extension with mass spectrometry detection, pyrosequencing, the 5' nuclease assay and the Invader assay, all suffer from the limitation that they require specialized instrumentation. Commercially available products like SNaPshot (Applied BioSystems, Foster City, Calif.) and SNuPe (Amersham Pharmacia Biosciences, Piscataway, N.J.) make use of popular DNA sequencers which are expensive instruments to install and maintain. The 5 ' nuclease assay, such as TaqMan assay, requires the use of expensive preparation of fluorescent- labelled probe for each allele.

The biochemistry of allele discrimination includes three categories: discrimination based on the properties of DNA polymerases, the properties of DNA ligases, and the properties DNA hybridization. Of these three, methods based on the properties of DNA polymerases are the most popular. Several properties of DNA polymerases have been exploited for SNP (single nucleotide polymorphism) genotyping, primer extension being a popular example. Technically, primer extension can be performed in two ways: one is to anneal an extension primer immediately upstream to the target polymorphism; the other is to design allele specific extension primers with the 3' base matching the polymorphic target. The former approach identifies polymorphism by identifying the bases extended. Since the identification of the target polymorphisms needs only one base extension, this approach is known as single base extension (SBE) or mini-sequencing. The latter approach infers the polymorphism by detecting the products of extension from the allele specific primers. Allele- specific polymerase chain reaction (AS-PCR) is based on this principle (FIG. 2). It is a useful technique that has been exploited for SNP genotyping by several groups. Compared to popular SBE, AS-PCR has certain advantages. For example, it is a single step reaction, DNA amplification and allele discrimination are combined together, and its products are suitable for analysis by DNA sequencers or other instruments. Unfortunately, AS-PCR also has certain limitations, e.g. some SNPs may not be amenable to AS-PCR and their allele discrimination might not be optimal. Part of the problem originates from the 3' mismatch bases of the allele specific primers. For some SNP markers, the mismatch of 3' allele specific bases may not be sufficient to block the extension of DNA polymerases, making it difficult to distinguish the two alleles. However, when AS-PCR is performed and assayed kinetically, a clear difference between the matched and mismatched primers and alleles can be identified reliably. The different outcomes from end-point and kinetic assays suggests that multiple cycles of thermal amplification tend to blur the distinctions. While it is possible to limit the number of cycles to achieve a clear difference between matched and mismatched primers, it is difficult to optimize the number of cycles because factors, such as variability of sample preparation, can affect the amplification efficiency. Recently, the use of the locked nucleic acid (LNA) in oligonucleotides has been shown to improve the performance of allele- specific PCR, but it is expensive to manufacture such modified primers.

It would be highly desirable to have a method for high-throughput genotyping of this NC_000014.8:g.l05239192T>C polymorphism that is cost effective, fast, highly discriminating, exceptionally reliable, and readily amenable to analysis using common laboratory equipment.

SUMMARY OF THE INVENTION

The present invention provides a new allele specific PCR (AS-PCR) design, that utilizes widely available real time PCR machines, to identify the AKT1 rs2494732 polymorphism (c.l l72+23A>G or NC_000014.8:g.l05239192T>C). The design couples two separate AS-PCR reactions with allele- specific primer for each PCR reaction in the presence of a DNA binding dye and a thermostable DNA polymerase enzyme having 3'— >5' exonuclease activity. In particular, the third nucleotide from the 3' end of the allele-specific primer is changed to a different nucleotide from the normal sequence to enhance discrimination of the two alleles during PCR amplification. The threshold cycle (Ct) of each AS-PCR reaction is measured using a real time PCR machine and the difference in threshold cycle (Ct) is used to deduce the genotype of the individual. The present invention is particularly useful for rapid determination of the genotype for this AKT1 rs2494732 polymorphism.

In another embodiment, the present invention includes a method for amplifying the two alleles, wherein the method comprises: preparing a reaction mixture comprising: (i) a DNA sample comprising either or both alleles, wherein the two alleles differ by a single nucleotide polymorphism; (ii) an allele-specific extension primer having a sequence complementary at its 3' nucleotide to the target allele; (iii) a common extension primer having a sequence complementary to a remote locus common to both polynucleotide templates of the target allele and the variant allele, and having the opposite orientation relative to the allele-specific extension primer; (iv) a dNTP mixture; and (v) a thermostable DNA polymerase enzyme having 3'→5' exonuclease activity; and then amplifying the polynucleotide segment under polymerase chain reaction conditions, wherein the annealing temperature and Mg2+ ion concentration are optimized to selectively effect hybridization of the extension primer to the correct target allele (FIG. 4). The present invention also includes a kit for determining the single nucleotide polymorphism in a DNA sample wherein the kit comprises: (a) an allele-specific extension primer having a sequence complementary at its 3' nucleotide to the target allele; (b) a common extension primer having a sequence complementary to a remote locus common to both alleles, wherein the common extension primer has the opposite orientation relative to the allele-specific extension primer; (c) a dNTP mixture; (d) a fluorescent double stranded DNA binding dye; and (e) a polymerase enzyme having 3'→5' exonuclease.

The present invention further includes a kit comprising of the kit necessary to determine the single nucleotide polymorphism, the packaging materials; instructions for using the components, performing the real time PCR reaction, and interpreting the data; one or more containers for holding the components; standards for calibrating the real time allele- specific PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing the rs24947732 single nucleotide polymorphism that creates a Pst I restriction enzyme recognition site.

FIG. 2 is a schematic representation of the allele-specific PCR using an allele- specific oligonucleotide primer to each target variant and a common primer.

FIG. 3 is showing representative examples of the allele-specific real time PCR results for different genotypes.

FIG. 4 is showing the melting curve analysis of the PCR product from the allele- specific PCR amplification.

FIG. 5 is a flowchart showing steps of the exemplary method of the invention.

FIG. 6 is a diagrammatic view of an exemplary kit of the invention.

DETAILED DESCRIPTION

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Definitions

Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Numeric ranges recited herein are inclusive of the numbers defining the range and include and are supportive of each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUBMB Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise noted, the terms "a" or "an" are to be construed as meaning "at least one of." The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the case of any amino acid or nucleic sequence discrepancy within the application, the figures control.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

As used herein, a "sample" refers to any substance containing or presumed to contain nucleic acid and includes a sample of tissue or fluid isolated from an individual or individuals, including but not limited to, for example, skin, check cell (e.g., by buccal swab), plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, tumors, and also to samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, recombinant cells and cell components).

As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to primers, probes, oligomer fragments to be detected, oligomer controls and unlabeled blocking oligomers and shall be generic to polydeoxyribonucleotides (containing 2-deoxy- D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. There is no intended distinction in length between the term "nucleic acid", "polynucleotide" and "oligonucleotide", and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single- stranded DNA, as well as double- and single-stranded RNA. The oligonucleotide is comprised of a sequence of approximately at least 6 nucleotides, preferably at least about 10-12 nucleotides, and more preferably at least about 15-25 nucleotides corresponding to a region of the designated nucleotide sequence. "Corresponding" means identical to or complementary to the designated sequence. The oligonucleotide is not necessarily physically derived from any existing or natural sequence but may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription or a combination thereof. The terms "oligonucleotide" or "nucleic acid" intend a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) is not found in nature.

Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends.

When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3' end of one oligonucleotide points toward the 5' end of the other, the former may be called the "upstream" oligonucleotide and the latter the "downstream" oligonucleotide.

The term "gene," as used herein, means a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and determines a particular characteristic in an organism.

The term "genetic locus," as used herein, means a specific position or specific region on a chromosome. In the context of the present invention, the term genetic locus refers to a particular position or region of polynucleotide sequences of a chromosome with which are associated multiple allelic variants.

The term "remote locus," as used herein, means either a locus which is upstream or downstream from the locus of a particular reference polynucleotide sequence.

The term "encompass," as used herein in reference to the location of a primer relative to a reference genetic locus, means that a PCR extension primer is located so as to amplify the genetic locus. The primer may include nucleotide sequences that correspond or are complementary to all or part of the genetic locus. Alternatively, the primer may be complementary to a region located 3' of the reference genetic locus.

The term "heterozygous," as used herein, means that a particular chromosomal loci has two or more different alleles. With reference to a sample, the term "heterozygous" means that the sample has two copies of a chromosome or polynucleotide sequence that have two or more different alleles at a particular locus.

The term "amplification," as used herein means the exponential reproduction of a polynucleotide sequence under conditions of polymerase chain reaction (PCR).

The term "primer" may refer to more than one primer and refers to an oligonucleotide, whether occurring naturally, as in a purified restriction digest, or produced synthetically, which is capable of acting as a point of initiation of synthesis along a complementary strand when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is catalyzed. Such conditions include the presence of four different deoxyribonucleoside triphosphates and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer ("buffer" includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature.

The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 nucleotides, although it may contain more or fewer nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

The term "extension primer," as used herein, means a polynucleotide sequence that is complementary to a template sequence, and which is capable of hybridizing and extending a sequence under polymerase chain reaction conditions.

The term "common primer," as used herein, means a polynucleotide sequence that is complementary to a region that is common to two or more polynucleotide templates. The term "allele," as used herein, means a particular genetic variant or polymorphism in the sequence of a gene, representing an alternative form of the gene.

The term "target allele" or "target sequence," as used herein, refers to a region of a polynucleotide template sequence that is to be selectively amplified. The target allele resides between the two primer sequences used for amplification. The target allele may represent either a wild-type or consensus sequence, characteristic of the predominant form of the gene, or alternatively may represent a polymorphic variant that is present in a population at a lower frequency.

The term "variant allele" or "variant sequence," as used herein, refers to a region of a polynucleotide template sequence that differs from the target allele by one or more nucleotides, and with respect to which the target allele is being selectively amplified. The variant allele may represent either a wild-type or consensus sequence, or alternatively may represent a polymorphic variant that is present in a population at a lower frequency. Thus, the terms "target allele" and "variant allele" are used arbitrarily to designate one allele and another allele, and are not used to designate or differentiate the frequency of any particular allele in a population.

The term "allele-specific," when used in reference to nucleic acid sequences, such as oligonucleotides and primers, means that the nucleic acid sequence is complementary with the target allele of a reference sequence. As used herein, an "allele-specific" primer is a primer that is exactly complementary to the nucleotides of the target allele that differ from the variant allele. For example, if a target allele and a variant allele differ by a single nucleotide polymorphism, the 3' end of the primer will be exactly complementary to one or the other nucleotide polymorphic variants (which will be the 5' nucleotide of the target allele). While the allele-specific primer is preferably exactly complementary to the target allele with respect to all nucleotides, it is contemplated that the method of the present invention also includes use of allele-specific primers that are exactly complementary at the 3' end of the primer, but are only substantially complementary at other nucleotides positions, such that the allele-specific primer preferentially hybridizes and extends under PCR conditions to the target allele relative to the variant allele.

The term "complement," and its related adjectival form "complementary," when used in reference to two nucleic acid sequences, means that when two nucleic acid sequences are aligned in anti-parallel association (with the 5' end of one sequence paired with the 3' end of the other sequence) the corresponding G and C nucleotide bases of the sequences are paired, and the corresponding A and T nucleotide bases are paired. Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine.

The primers herein are selected to be "substantially" complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non- complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Typically, the primers have exact complementarity to obtain the best detection results.

The term "Tm" means the melting temperature, or annealing temperature, of a nucleic acid duplex at which, under specified conditions, half of the base pairs have disassociated. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength, and incidence of mismatched base pairs. The "predicted Tm," as used herein, means the temperature at which a primer and its complementary template sequence are predicted to be sufficiently stable to permit hybridization and extension by PCR, and is determined using the nearest neighbor algorithm (Von-Ahsen N et al 1999 Clinical Chemistry, 45:12, 2094- 2101). A software tool for determining the predicted Tm for oligonucleotides and primers based on the nearest neighbor algorithm is available at http://www.idtdna.com.

As defined herein, "3'→5' exonuclease activity" refers to the activity of a template- specific nucleic acid polymerase having a 3'→5' exonuclease activity associated with some DNA polymerases, in which one or more nucleotides are removed from the 3' end of an oligonucleotide in a sequential manner.

A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR is conducted in a thermal cycler equipped with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase. The PCR process generally comprises of a series of repeated temperature changes. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the double-stranded DNA molecules; the second, at an optimized temperature for primer annealing; the third, at between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase.

A DNA-binding dye binds to all double- stranded DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR leads to an increase in fluorescence intensity measured at each cycle and is captured by a detector in the real time machine. However, DNA dyes such as SYBR Green will bind to all double- stranded PCR products, including nonspecific PCR products (such as primer-dimer). This can potentially interfere with, or prevent, accurate monitoring of the intended target sequence. In real-time PCR with DNA-binding dyes the reaction is prepared as usual, with the addition of fluorescent DNA- binding dye. Then the reaction is run in a real-time PCR instrument, and after each cycle, the intensity of fluorescence is measured. This method has the advantage of only needing a pair of primers to carry out the amplification, which keeps costs down; however, only one target sequence can be monitored in a tube and the PCR condition, such as magnesium concentration and primer annealing temperature must be optimized to avoid primer-dimer formation and non-specific amplification of other sequences.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA techniques, oligonucleotide synthesis which are within the skill of the art. Such techniques are explained fully in the literature. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal, 1984); and a series, Methods in Enzymology (Academic Press, Inc.), the contents of all of which are incorporated herein by reference.

Preparation of Polynucleotide Templates

Any polynucleotide molecule, in purified or non-purified form, can be utilized as the starting nucleic acid or acids, provided it contains the sequence being detected. Thus, the process may employ, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single-stranded or double-stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acids produced from a previous amplification reaction herein using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid.

The nucleic acid templates may be obtained from blood, tissue material such as chorionic villi or amniotic cells by a variety of techniques such as that described by Maniatis et al., Molecular Cloning (1982), 280-281. The method of the present invention is particularly useful in analyzing genomic DNA.

The cells may be directly used without purification of the nucleic acid if they are suspended in hypotonic buffer and heated to about 900-100° C, until cell lysis and dispersion of intracellular components occur, generally about 1 to 15 minutes. After the heating step the amplification reagents may be added directly to the lysed cells. This direct cell detection method may be used on peripheral blood lymphocytes and amniocytes.

The target nucleic acid contained in the sample may be in the form of genomic DNA, and then denatured, using any suitable denaturing method, including physical, chemical, or enzymatic means, which are known to those of skill in the art. A preferred physical means for strand separation involves heating the nucleic acid until it is completely (>99%) denatured. Typical heat denaturation involves temperatures ranging from about 80° C. to about 105° C, for times ranging from a few seconds to minutes.

The denatured nucleic acid strands are then incubated with preselected oligonucleotide primers under conditions that facilitate the binding of the primers to the single nucleic acid strands. As known in the art, the primers are selected so that their relative positions along a duplex sequence are such that an extension product synthesized from one primer, when the extension product is separated from its template (complement), serves as a template for the extension of the other primer to yield a replicate chain of defined length.

In one embodiment, the allele-specific PCR amplification method of the present invention is used to selectively amplify a polynucleotide segment of a gene in a sample that has a known polymorphic variant or allele. In this case, the sample material will comprise polynucleotide templates that may have only one allele or both alleles.

Real Time Allele- Specific PCR Amplification

The present invention contemplates use of the PCR-based method for determining the presence or absence of a specific known nucleic acid sequence, such as a mutation (e.g. a genetic polymorphism), called allele-specific PCR (ASP) or allele-specific amplification (ASA), also known as amplification refractory mutation system (ARMS) and PCR amplification of specific alleles (PASA), as described in U.S. Pat. No. 5,639,611; Ruano et al., Nucleic Acids Res 17:8392 (1989) (allele-specific amplification), Ruano et al., Nucleic Acids Res. 19:5887-5882 (1991) (coupled amplification and sequencing) and Cheng et al., Nature 368:664-665 (1994). Allele-specific PCR amplification (ASA) is used to selectively amplify one specific predetermined allele from a sample containing multiple alleles at the same genetic locus.

In a typical ASP assay one or more PCR reactions using different PCR extension primers are annealed to the same nucleic acid sample. The PCR primers are designed to have a residue at the 3 '-terminus of the primer (complementary to the 5' primer initiation site of the template) that is complementary to one of the two allelic variants and not to the other. The PCR reaction does not extend from a primer having a 3 '-terminal mismatched base, unless the polymerase used has a 3'→5' proofreading activity that removes the mismatched base and inserts the correct base. Proofreading repairs the PCR primer and destroys the extension discrimination between the two alleles. Therefore, a polymerase lacking 3'→5' proofreading activity, such as Taq DNA polymerase has traditionally been used in an ASP assay.

Although discrimination between specificity of PCR extension from the allele- specific ASP primers is known to be enhanced by the introduction of deliberate multiple mismatches near the 3 '-terminal nucleotide, this significantly reduces the overall PCR extension product yield. See, e.g., Ruano et al., Nucleic Acids Res. 17:8392 (1989). Other factors known to affect the stability of the hybridization of PCR primers in an ARMS assay include the position of additional mismatches in the primer, the GC content of the 5 or 6 nucleotides preceding the 3' nucleotide, and the discriminatory 3 '-terminal nucleotide, depending on the difference between the alleles and the type of mismatch. The destabilization is greater when the second mismatch is nearer to the 3 '-terminal nucleotide. The destabilizing effect of additional mismatches on ASP has been ranked qualitatively (CC>CT>GG=AA=AC>GT).

The present invention is directed to a method particularly optimized for rapid detection of the AKT1 rs2494732 polymorphism (c.ll72+23A>G or NC_000014.8:g.l05239192T>C). The discriminatory capability of the method of the present invention is further enhanced by converting the third nucleotide from the 3' end of the allele- specific extension primers. To overcome the low overall PCR extension yield as a result of introducing a mismatch at this particular position, the PCR amplification is monitored with the introduction of a fluorescent DNA-binding dye that gives a high sensitivity of detection of the PCR extension product. The primer sequences and the Tm annealing temperature are also optimized to avoid primer-dimer formation or non-specific amplification of other sequences which may interfere with the interpretation of the final results.

To briefly summarize, in the first step of the reaction, the nucleic acid molecules of the sample are transiently heated, and then cooled, in order to denature double- stranded molecules. Forward and reverse primers are present in the amplification reaction mixture at an excess concentration relative to the sample target. When the sample is incubated under conditions conducive to hybridization and polymerization, the primers hybridize to the complementary strand of the nucleic acid molecule at a position 3' to the sequence of the region desired to be amplified that is the complement of the sequence whose amplification is desired. Upon hybridization, the 3' ends of the primers are extended by the polymerase. The extension of the primer results in the synthesis of a DNA molecule having the exact sequence of the complement of the desired nucleic acid sample target. The PCR reaction is capable of exponentially amplifying the desired nucleic acid sequences, with a near doubling of the number of molecules having the desired sequence in each cycle. Thus, by permitting cycles of hybridization, polymerization, and denaturation, an exponential increase in the concentration of the desired nucleic acid molecule can be achieved. The fluorescent DNA binding dye which binds only to double-stranded DNA molecules is included in the reaction mixture. Upon binding to the double-stranded PCR DNA product, the dye can emit a characteristic light spectrum when it is excited by a laser beam. The light emission can be captured by a CCD camera installed inside a real time PCR machine.

EXAMPLES

Oligonucleotide Synthesis

Oligonucleotides were synthesized by IDT DNA Technologies, Inc., using their proprietary technologies but could easily obtained from other commercial vendors.

Source and isolation of human DNA

Genomic DNA samples were isolated from cancer cell lines using the Qiagen blood DNA minikit. The rs2494732 genotype of these cell lines was compared in parallel with PCR restriction fragment polymorphism (PCR-RFLP) technique. The A>G conversion created a Pst I restriction enzyme in the DNA that can be used to differentiate the polymorphism (FIG. 1).

PCR amplification

Allele-specific real time PCR reaction is set up as described in FIG. 5. Two separate allele-specific real time reactions were prepared and were run together on the same PCR plate using the Strategene MX3000P real time PCR machine. The forward allele-specific primers, AKT-T (5 ' -GGGGATGGAGGTGTAGCCCGT-3 ' ), which was used to detect the T-allele, and AKT-C (5 ' -GGGGATGGAGGTGTAGCCCGC-3 ' ) , which was used to detect the C-allele, were separately combined with the reverse common primer AKT-COM (5'- GCGGTCGCCTGCCCTTCTACAACC-3') in PCR reaction mix that contained 0.25 uM forward primer; 0.25 uM reverse primer; 2.5 mM Mg2Cl; 50 mM KC1; 10 mM Tris-HCl (pH 8.3); 5% DMSO (v/v), 0.2 mM each of dATP, dCTP, dGTP and dTTP; 6.25 uM SYTO 21; and 0.5 unit of Amplitaq Gold (ThermoFisher). Reactions were carried out for 95° C for 10 min, 40 amplification cycles at 95° C for 15s, 65° C for 30 s and 72° C for 30s. The CCD camera was set to capture the fluorescent signal during polymerization at 72° C. At the end of the PCR amplification, a melting curve analysis was performed by heating the PCR extension product to 95° C for 1 min and then cooling to 55° C for 1 min before heating up to 95° C again at a rate of 0.3° C per second. The fluorescent signal was captured during the heating up of the PCR extension product from 55° C to 95° C. The allele-specific PCR amplification had been conducted at three separate primer annealing temperatures at Mg2+ concentration of 2.5 mM to identify the optimal annealing temperature for the amplification. Non-specific amplification products or primer-dimers were detected at both 55° C and 60° C but not at 65° C (Fig. 4).

Genotype determination

The threshold cycles (Ct) of two separate allele-specific real time reactions were determined by the real time PCR machine (FIG. 3). Panel A shows the real time PCR curves for homozygous normal, heterozygous carrier and homozygous affected. In the detection of rs24947732 polymorphism, when an individual is homozygous for the T allele, i.e. T/T, there is a wide separation between the T-allele amplification curve and the C-allele amplification curve. The separation can be represented by ACt, i.e. subtracting the Ct value of the T-allele amplification curve from that of the C-allele amplification curve. When an individual is homozygous for the C allele, i.e. C/C, the ACt value will decrease to a negative value. The ACt values were determined and matched with their genotypes. A genotype of T/T, T/C and C/C were concluded if ACt was >10, -5< ACt >5, and <-10 respectively. Panel B shows that several cell line DNAs had been genotyped by this PCR assay and their genotypes confirmed by the PCR-RFLP with Pst I restriction enzyme (Fig. 1).

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for conducting an allele-specific real time polymerase chain reaction to detect a nucleotide polymorphism, NC_000014.8:g.l05239192T>C, of the v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1) gene, comprising:

a. obtaining a genomic DNA sample from a test subject;

b. subjecting said genomic DNA sample to two separate polymerase chain reactions;

c. utilizing in said one of the polymerase chain reactions with a primer specific to direct amplification of the T-allele with the following sequence:

SEQ1: T- Allele 5 ' -GGGGATGGAGGTGTAGCCCGT-3 ' ;

d. utilizing in said the other polymerase chain reactions with a primer specific to direct amplification of the C-allele with the following sequence:

SEQ2: C-Allele: 5 ' -GGGGATGGAGGTGTAGCCCGC-3 ' ;

e. utilizing in said both polymerase chain reactions with a primer to direct amplification of either alleles with the following sequence:

SEQ3: 5'-GCGGTCGCCTGCCCTTCTACAACC-3'; and

f. utilizing a composition for amplifying and monitoring a target nucleic acid sequence in the sample.

2. The method of claim 1 wherein the composition for amplifying and monitoring a target nucleic acid sequence in a sample comprises a thermostable DNA polymerase, deoxynucleoside-tri-phosphates, a fluorescent double-stranded DNA-binding dye, and a buffer containing Mg2+ ions.

3. The method of claim 1 wherein the third nucleotide from 3' end of SEQ1 consists one of cytosine, adenosine or guanine.

4. The method of claim 1 wherein the third nucleotide from 3 ' end of SEQ2 consists one of cytosine, adenosine or guanine.

5. The method of claim 1 wherein the two separate polymerase chain reactions are performed simultaneously by real time PCR.

6. The method of claim 5 wherein the threshold cycle (Ct) values of the two separate polymerase chain reactions are determined.

7. The method of claim 6 wherein the difference between the Ct values of the two separate polymerase chain reactions are determined.

8. The method of claim 7 wherein the Ct value difference is used to determine the genotype of the sample.

9. The method of claim 1 wherein the genomic DNA specimen comprises at least one of cells from a buccal swab, saliva, hair, and venous blood.

10. A kit for determining the NC_000014.8:g.l05239192T>C nucleotide polymorphism in a DNA sample wherein the kit comprises:

a. an allele-specific extension primer having a sequence complementary at its 3' nucleotide to the T-allele wherein the third nucleotide from 3' end of the primer consists one of cytosine, adenosine or guanine;

b. an allele-specific extension primer having a sequence complementary at its 3' nucleotide to the C-allele wherein the third nucleotide from 3' end of the primer consists one of cytosine, adenosine or guanine;

c. a primer to direct amplification of either alleles;

d. a thermostable DNA polymerase;

e. deoxynucleoside-tri-phosphates,

f. a fluorescent double stranded DNA-binding dye, and

g. a buffer.

11. A kit for determining the NC_000014.8:g.l05239192T>C nucleotide polymorphism comprising:

a. the kit of claim 10;

b. packaging materials;

c. instructions for using the components, performing the real time PCR reaction, and interpreting the data;

d. one or more containers for holding the components, and

e. standards for calibrating the real time allele-specific PCR.