Method and kits for detection of fragile X specific, GC-rich DNA sequences

A method is provided for amplifying and detecting specific GC-rich nucleic acid sequences contained in a nucleic acid or in a mixture of nucleic acids, which includes treating a separate nucleic acid containing the specific sequence with a molar excess of primers and a polymerase and extending the primers in the presence of dATP, dCTP, TTP, and an analogue of dGTP. In one application of the present invention, individuals who are carriers for, or afflicted by, the fragile X syndrome are detected.

FIELD OF THE INVENTION
 This invention relates generally to methods for amplifying, detecting and
 cloning nucleic acid sequences, particularly to methods that are based on
 a polymerase chain reaction (PCR). It relates to a process for amplifying
 nucleic acid sequences in a test sample. One can also employ the present
 invention to determine whether a specific nucleic acid sequence is present
 in a test sample. More specifically, this invention relates to a process
 for amplifying a selected GC-rich nucleic acid sequence to facilitate
 detection and/or cloning of the sequence. The process of the present
 invention uses, in one embodiment, a thermostable polymerase to catalyze
 the extension of a primer bound to a template. The present invention also
 relates to the development of diagnostic assays for inherited or sporadic
 genetic defects. One application of the present invention provides an
 assay for the genetic defect that causes the fragile X syndrome in
 carriers, persons afflicted, fetuses and embryos.
 BACKGROUND
 The fragile X syndrome is the most common inherited form of mental
 retardation and developmental disability. This condition afflicts
 approximately 1 in 1250 males and 1 in 2000 females.
 As the name implies, fragile X is an X chromosome-linked condition. The
 fragile X phenotype is characterized by a visible constriction near the
 end of the X chromosome, at locus q27.3, and there is a tendency for the
 tip of the X-chromosome to break off under certain conditions in tissue
 culture. These tissue culture procedures form the basis of the assay most
 commonly used for fragile X at present.
 The pattern of inheritance of this condition is atypical of that associated
 with X-linked conditions. Typically, there is a 50% probability that the
 son of a woman who carries an X-linked genetic defect will be afflicted by
 the defect. Additionally, all males who carry the abnormal gene are
 afflicted by the X-linked condition in the typical pattern. Furthermore,
 since females have two X chromosomes, they normally do not suffer the
 effects of a single damaged X chromosome.
 In fragile X, however, some carrier males are phenotypically normal.
 Moreover, about one third of the females who inherit the fragile X
 chromosome are afflicted. The incidence of carrier males in different
 generations of a family varies. Daughters of carrier males are generally
 non-expressing carriers, but may have afflicted sons. Furthermore,
 afflicted daughters occur more frequently among the offspring of carrier
 mothers than among the offspring of carrier fathers (Brown, The Fragile X:
 Progress toward Solving the Puzzle, Am. J. Human Genet. 47 175-80, 1990).
 Researchers recently identified the genomic region associated with this
 condition. (Oberle, et al., Instability of a 550-Base Pair DNA Segment and
 Abnormal Methylation in Fragile X Syndrome, Science 252 1097-1102, 1991;
 Kremer, et al., Mapping of DNA Instability at the Fragile X to a
 Trinucleotide Repeat Sequence p(CCG)n, Science 252 1711-14, 1991; and
 Bell, et al., Physical Mapping across the Fragile X Hypermethylation and
 Clinical Expression of the Fragile X Syndrome, Cell 64 861-66, 1991).
 Additionally, researchers have sequenced a partial cDNA clone derived from
 this region, called FMR-I. (Verkerk, et al., Identification of a Gene
 (FMR-1) Containing a CGG Repeat Coincident with a Breakpoint Cluster
 Region Exhibiting Length Variation in Fragile X Syndrome, Cell 65 905-14,
 1991). The Oberle, Kremer, Bell and Verkerk papers are hereby incorporated
 by reference.
 These studies provide an explanation for the atypical pattern of
 inheritance of fragile X. The mutation that ultimately results in the
 fragile X phenotype occurs in stages. In the early stages, the gene is not
 fully defective, rather there is a "pre-mutation" of the gene. Carriers of
 the premutation have a normal phenotype. A further mutation occurs in
 carrier females that produces the phenotype in their offspring.
 The coding sequence for FMR-I contains a variable number of CGG repeats.
 Individuals who are not carriers have approximately 30 CGG repeats in
 their FMR-I. Carriers, however, have between 50 and 200 CGG repeats. This
 amplification of the FMR-I CGG sequence is the pre-mutation. Afflicted
 individuals have even more CGG repeats. As many as several thousand CGG
 repeats have been observed in afflicted individuals. (Oberle, et al.,
 1991).
 However, most affected individuals do not express the FMR-1 mRNA (Pieretti,
 et al., Absence of Expression of the FMR-1 Gene in Fragile X Syndrome,
 Cell 66 1-201991). A CpG island, located upstream of the CGG repeat
 region, is methylated when the number of CGG repeats is above a threshold
 of about 200 copies (Oberle, et al., 1991; Kremer, et al., 1991, Bell, et
 al., 1991). This methylation inactivates the gene.
 Until now, the only way to diagnose the fragile X syndrome has been to
 examine microscopically an afflicted individual's chromosomes after cell
 growth and treatment in tissue culture. In such an examination, the
 laboratory examined the X chromosome to ascertain whether it was
 characteristically constricted, or had a broken tip. This method is both
 costly and not reliable. For example, this method misses almost all male
 carriers and half of the female carriers of the fragile X syndrome. (THE
 FRAGILE X SYNDROME, Oxford Univ. Press (Davies, ed. 1989)) Another method
 for detecting fragile X carriers and genotypes employs a Southern blot
 methodology but lacks sensitivity and speed. (Rousseau et al. Direct
 Diagnosis by DNA Analysis of the Fragile X Syndrome of Mental Retardation,
 N.E. J. Med. 1673-81 (1991))
 The present invention provides a fast, inexpensive genetic test for
 reliably identifying carriers of the fragile X genotype based on molecular
 structure of the gene defect. The method of the present invention
 determines whether the number of CGG repeats in the test individual's
 X-chromosome are characteristic of a normal, carrier or afflicted person.
 The test method of the present invention is based on the polymerase chain
 reaction (PCR). PCR-based assays are ideal for detecting specific DNA
 sequences that are present in low abundance relative to the total DNA. In
 brief, a PCR method amplifies the specific DNA sequence, for example, one
 hundred thousand to a million fold. Once amplified to this level, the
 specific DNA sequence, if present, is readily detected.
 Prior attempts to develop PCR-based methods to directly identify the CGG
 repeat sequence at the genomic level have been unsuccessful (Kremer, et
 al., 1991), or only partially successful (Fu et al. Variations of the CGG
 Repeat at the Fragile X Site Results in Genetic Instability: Resolution of
 the Sherman Paradox, Cell 67: 1047-58 (1991)). This region appears
 unstable and difficult to clone or to analyze directly.
 The inability of PCR-based methods to detect GC-rich sequences has hindered
 the development of an assay for other conditions. For example, clonality
 in Epstein-Barr virus infection, the androgen receptor gene, the
 beta-adrenergic receptor and the CMV genome are each characterized by a
 GC-rich nucleic acid sequence. It has not been possible to identify
 clonality of the Epstein-Barr virus with conventional PCR methods.
 Moreover, as the androgen receptor has a CAG repeat region, the
 beta-adrenergic receptor has an 80% GC rich region and the CMV genome has
 portions that are more than 75% GC, none of these nucleic acids are
 amplifiable by conventional PCR methods.
 We have solved the problem of using PCR-based methods with GC-rich nucleic
 acid sequences. Using our method, we have amplified and detected the
 GC-rich region of the FMR-1 gene in normals, carriers and afflicted
 individuals.
 SUMMARY OF THE INVENTION
 The present invention amplifies selected GC-rich nucleic acid sequences
 present in a test sample. In one embodiment of the present invention,
 7-deaza dGTP was substituted for dGTP.
 The present invention can be used to assay for the GC-rich nucleic acid
 sequence characteristic of carriers of, and persons afflicted with, the
 fragile X syndrome.

DETAILED DESCRIPTION OF THE INVENTION
 U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159 and 4,965,188, which are
 hereby incorporated by reference, provide additional details of the PCR
 process which is modified by the present invention.
 Oligonucleotide such as those comprised of two or more deoxyribonucleotides
 or ribonucleotides, preferably more than three are useful in the practice
 of the present invention. An oligonucleotide's size and sequence
 determines its function or use. An oligonucleotide may be derived
 synthetically or by cloning.
 Primer useful in the present invention include oligonucleotides capable of
 acting as a point of initiation of DNA or RNA synthesis. A primer may be
 purified from a restriction digest by conventional methods, or it may be
 produced synthetically.
 PCR typically employs two primers that bind to a selected nucleic acid
 template. The primers are combined with the other PCR reagents under
 conditions that induce primer extension, i.e., with four different
 nucleoside triphosphates (or analogues thereof), an appropriate polymerase
 and an appropriate buffer ("buffer" includes pH, ionic strength,
 cofactors, etc.) at a suitable temperature. In a PCR method where the
 polymerase is Taq polymerase, the buffer preferably contains 1.5-2 mM of a
 magnesium salt, preferably MgCl.sub.2, 150-200 .mu.M of each nucleoside,
 triphosphate (or analog thereof), 1 .mu.M of each primer, preferably with
 50 mM KCl, 10 mM Tris buffer at pH 8.4, and 100 .mu.g/ml gelatin.
 The primer is preferably single stranded for maximum efficiency in
 amplification, but it may be double stranded. Double stranded primers are
 first "denatured", i.e. treated to separate its strands before being used
 to prepare extension products. A preferred means of denaturing double
 stranded nucleic acids is by heating.
 In the present invention, a primer must be sufficiently long to "prime" the
 synthesis of extension products in the presence of an appropriate
 polymerase and other reagents. The primer length depends on many factors,
 including the temperature and source of the primer and the use of the
 method. Typically, in the practice of the present invention, the primer
 contains 15-25 or more nucleotide residues. Short primer molecules
 generally require lower reaction temperatures to form and maintain the
 primer-template complexes which support the chain extension reaction.
 The primers used in the present method are "substantially" complementary to
 a nucleic acid containing the selected sequence to be amplified, i.e. the
 primers must bind to, or hybridize with, a nucleic acid containing the
 selected sequence (or its complement). Nonetheless, the primer sequence
 need not be an exact complement 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 nucleic acid containing the selected sequence. Alternatively, one or
 more non-complementary bases can be interspersed into the primer, provided
 that the primer sequence has sufficient complementarity with the sequence
 of the nucleic acid containing the selected sequence to (i) hybridize
 therewith and (ii) support a chain extension reaction. Notwithstanding the
 above, primers which are fully complementary to the nucleic acid
 containing the selected sequence are preferred to obtain the best results.
 Any specific nucleic acid sequence can be produced by the present process.
 It is only necessary that a sufficient number of bases at both ends of the
 sequence be known in sufficient detail so that two oligonucleotide primers
 can be prepared which will hybridize to different strands of the desired
 sequence and at relative positions along the sequence such that an
 extension product synthesized from one primer, when it is separated from
 its template (complement), can serve as a template for extension of the
 other primer into a nucleic acid of defined length. The greater the
 knowledge about the bases at both ends of the sequence, the greater can be
 the specificity of the primers for the target nucleic acid sequence, and
 thus the greater the efficiency of the process.
 In a preferred embodiment of the present invention, two primers are used.
 One of the primers is complementary to (i) a sequence at the 3' end of the
 selected sequence, (ii) a sequence which abuts, or is near the 3' end of
 the selected sequence, or (iii) a sequence which includes the 3' end of
 the selected sequence as well as a sequence which abuts the 3' end of the
 selected sequence. The other primer, in this preferred embodiment,
 contains (i) the sequence at the 5' end of the selected sequence (ii) a
 sequence which abuts or is near the 5' end of the selected sequence or
 (iii) the sequence at the 5' end of the selected sequence as well as the
 sequence which abuts the 5' end of the selected sequence. Alternatively,
 either primer may be replaced by a primer which binds to, or hybridizes
 with, the complement of any of the foregoing preferred primers.
 The terms "restriction endonucleases" and "restriction enzymes" refer to
 enzymes, commonly from bacteria, that cut double-stranded DNA at or near a
 specific nucleotide sequence.
 The term "thermostable enzyme" refers to a polymerase which is heat stable
 and resistant and which catalyzes the formation of primer extension
 products complementary to a template. Generally, the synthesis will be
 initiated at the 3' end of each primer and will proceed in the 5' to 3'
 direction along the template strand, until synthesis terminates.
 Theoretically, this process produces DNA or RNA copies of different
 lengths. There may be thermostable enzymes which are useful in the present
 invention, which initiate synthesis at the 5' end and proceed in the other
 direction, using the process described above.
 The thermostable enzyme herein must satisfy a single criterion to be
 effective for the amplification reaction of this invention, i.e., the
 enzyme must not become irreversibly denatured (inactivated) when subjected
 to the elevated temperatures for the time necessary to effect denaturation
 of double-stranded nucleic acids. Irreversible denaturation for purposes
 herein refers to permanent and complete loss of enzymatic activity. The
 heating conditions necessary for denaturation will depend, e.g., on the
 buffer salt concentration and the length and nucleotide composition of the
 nucleic acids being denatured, but typically range from about 90.degree.
 C. to about 105.degree. C. for a time depending mainly on the temperature
 and the nucleic acid length, typically about one half to four minutes.
 Higher temperatures may be tolerated as the buffer salt concentration
 and/or GC composition of the nucleic acid is increased. Preferably, the
 enzyme will not become irreversibly denatured at about 90-100.degree. C.
 The thermostable enzyme herein preferably has an optimum temperature at
 which it functions which is higher than about 40.degree. C., which is the
 temperature below which hybridization of primer to template is promoted,
 although, depending on (1) the concentration of magnesium and other salts
 in the buffer and (2) the composition and the length of the primer,
 hybridization can occur at higher temperatures (e.g., 45-70.degree. C.).
 The higher the enzyme's optimum temperature, the more specific and/or
 selective the primer-directed extension process is. However, enzymes that
 are active below 40.degree. C., e.g., at 37.degree. C., are also within
 the scope of this invention. Preferably, the optimum temperature ranges
 from about 50 to 80.degree. C., more preferably above about 60.degree. C.
 Examples of enzymes which have been reported in the literature as being
 resistant to heat include heat-stable polymerases, such as, e.g.,
 polymerases extracted from the thermostable bacteria Thermus flavus,
 Thermus ruber, Thermus thermophilus, Bacillus stearothermophilus (which
 has a somewhat lower temperature optimum than the others listed), Thermus
 aquaticus, Thermus lacteus, Thermus rubens, and Methanothermus fervidus.
 Other useful polymerases are those which can withstand repeated cycles in
 which DNA is denatured and then annealed with a primer by means other than
 heat denaturation. Nonetheless, unstable polymerases can be employed where
 additional enzyme is added with each cycle.
 The present invention is directed to a process for amplifying selected
 nucleic acid sequences. Because large amounts of the selected sequence may
 be produced by this process, the present invention may be used for
 improving the efficiency of cloning DNA or messenger RNA and for
 amplifying a selected sequence to facilitate detection thereof.
 In general, the present process involves a chain reaction for producing, in
 exponential quantities relative to the number of reaction steps involved,
 at least one selected nucleic acid sequence given (a) that the ends of the
 selected sequence are known in sufficient detail that oligonucleotides can
 be synthesized which will hybridize to them, and (b) that a small amount
 of the sequence is available to initiate the chain reaction. The product
 of the chain reaction will be a discrete nucleic acid duplex with termini
 corresponding to the ends of the primers employed.
 Any nucleic acid, in purified or non-purified form, can be utilized as the
 starting material. However, if the sample lacks the selected sequence, the
 process should not amplify any sequence. 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 selected nucleic acid sequence to be amplified may
 be only a fraction of a larger molecule, or it may be present initially as
 a discrete molecule where the selected sequence constitutes the entire
 nucleic acid.
 The selected sequence need not be purified; it may be a minor fraction of a
 complex mixture, such as a portion of the FMR-1 gene contained in human
 genomic DNA. The starting nucleic acid may contain two or more selected
 nucleic acid sequences, which may be the same or different. Therefore, the
 present process is useful not only for producing large amounts of one
 specific nucleic acid sequence, but also for amplifying simultaneously two
 or more selected nucleic acid sequences located on the same or different
 nucleic acid molecules.
 The nucleic acid or acids may be obtained from any source, for example,
 from plasmids, from cloned DNA or RNA, or from natural DNA or RNA from any
 source, including bacteria, yeast, viruses, organelles, and higher
 organisms such as plants or animals. DNA or RNA may be extracted from any
 nucleic acid containing sample such as 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: A Laboratory Manual
 (1982), 280-281.
 For the process using sequence-specific probes to detect the amplified
 material, the cells may be directly used without purification of the
 nucleic acid. For example, a cellular sample can be suspended in hypotonic
 buffer and heated to about 90-100.degree. C., until cell lysis and
 dispersion of intracellular components occurs. Such a process generally
 takes from about 1 to 15 minutes. After the heating step, the
 amplification reagents may be added directly to the lysed cells.
 If the nucleic acid contains two strands, it is necessary to separate the
 strands of the nucleic acid before it can be used as the template. This
 strand separation can be accomplished by any suitable denaturing method
 including physical, chemical or enzymatic means. One preferred physical
 method of separating the strands of the nucleic acid involves heating the
 nucleic acid until it is completely (&gt;99%) denatured. Typical heat
 denaturation involves temperatures ranging from about 90 to 105.degree. C.
 for times generally ranging from about 0.5 to 5 minutes. Preferably the
 effective denaturing temperature is about 90-100.degree. C. for about 0.5
 to 3 minutes. Strand separation may also be induced by an enzyme from the
 class of enzymes known as helicases or the enzyme RecA, which has helicase
 activity and in the presence of riboATP is known to denature DNA. The
 reaction conditions suitable for separating the strands of nucleic acids
 with helicases are described by Kuhn Hoffmann-Berling, CSH-Quantitative
 Biology, 43:63 (1978), and techniques for using RecA are reviewed in C.
 Radding, Ann. Rev. Genetics., 16:405-37 (1982). The denaturation produces
 two separated complementary strands of equal or unequal length.
 If the double-stranded nucleic acid is denatured by heat, the reaction
 mixture is allowed to cool to a temperature which promotes hybridization
 of each primer present to its complementary target (template) sequence.
 This temperature is usually from about 35.degree. C. to about 65.degree.
 C. or higher, depending on reagents, preferably from about 37.degree. C.
 to about 60.degree. C., maintained for a time effective to denature the
 double-stranded nucleic acid, generally from about 0.5 to 5 minutes, and
 preferably about 1-3 minutes. In practical terms, the temperature is
 simply lowered from about 95.degree. C. to about 65.degree. C. or to as
 low as about 37.degree. C., preferably to about 45-58.degree. C. for Taq
 polymerase, and hybridization occurs at a temperature within this range.
 Whether the nucleic acid is single- or double-stranded, the thermostable
 enzyme may be added at the denaturation step or when the temperature is
 being reduced to or is in the range for promoting hybridization. The
 reaction mixture is then heated to a temperature at which the activity of
 the enzyme is promoted or optimized, i.e., a temperature sufficient to
 increase the activity of the enzyme in facilitating synthesis of the
 primer extension products from the hybridized primer and template. The
 temperature must actually be sufficient to synthesize an extension product
 of each primer which is complementary to each nucleic acid template, but
 must not be so high as to denature each extension product from its
 complementary template (i.e., the temperature is generally less than about
 80.degree. C. to 90.degree. C.).
 Depending mainly on the types of enzyme and nucleic acid(s) employed, the
 typical temperature effective for this synthesis reaction generally ranges
 from about 40 to 80.degree. C., and preferably about 50 to 75.degree. C.
 The temperature more preferably ranges from about 65 to 75.degree. C. when
 a polymerase from Thermus aquaticus is employed. The period of time
 required for this synthesis may range from about 0.5 to 40 minutes or
 more, depending mainly on the temperature, the length of the nucleic
 acids, the enzyme and the complexity of the nucleic acid mixture,
 preferably about one to three minutes. If the nucleic acid is longer, a
 longer period of time is generally required.
 The newly synthesized strand and its complementary nucleic acid strand form
 a double-stranded molecule which is used in the succeeding steps of the
 process. In the next step, the strands of the double-stranded molecule are
 separated by heat denaturation at a temperature effective to denature the
 molecule, but not so high that the thermostable enzyme is completely and
 irreversibly denatured or inactivated. Depending mainly on the type of
 enzyme and the length of nucleic acid, this temperature generally ranges
 from about 90 to 105.degree. C., more preferably about 90 to 100.degree.
 C., and the time for denaturation typically ranges from about one half to
 four minutes, depending mainly on the temperature and the nucleic acid
 length.
 After this time, the temperature is decreased to a level which promotes
 hybridization (or annealing) of the primer to its complementary
 single-stranded molecule (template) produced from the previous step. Such
 temperature is described above.
 After this hybridization step, or in lieu of (or concurrently with) this
 hybridization step, the temperature is adjusted to a temperature which is
 effective to promote the activity of the thermostable enzyme to enable
 synthesis of a primer extension product using as a template the newly
 synthesized strand from the previous step. The temperature again must not
 be so high as to separate (denature) the extension product from its
 template, as previously described (usually from about 40 to 80.degree. C.
 for about 0.5 to 40 minutes, preferably about 50 to 70.degree. C. for
 about 1 to 3 minutes). Hybridization may occur during this step, so that
 the previous step of cooling before denaturation is not required. In such
 a case, using simultaneous steps, the preferred temperature range is
 between about 50 to 70.degree. C.
 The heating and cooling steps of strand separation, hybridization, and
 extension product synthesis can be repeated as often as needed to produce
 the desired quantity of the specific nucleic acid sequence, depending on
 the ultimate use. The only limitation is the amount of the primers, the
 thermostable enzyme and the nucleoside triphosphates present. Preferably,
 the steps are repeated at least once. For use in detection, the number of
 cycles will depend, e.g., on the nature of the sample. If the sample is a
 complex mixture of nucleic acids and the total nucleic acid is held
 constant, more cycles will be required to amplify the signal sufficiently
 for its detection. For general amplification and detection, preferably the
 process is repeated at least about 20 times.
 The process of the present invention uses an analogue of guanosine
 nucleotide. U.S. Pat. No. 4,804,748 discloses analogues useful in the
 present invention and is hereby incorporated by reference. Preferred
 analogues include inosine, 7-deaza-guanosine and 7-deaza inosine
 nucleotides (both ribo- and deoxyribo-). The 2'-deoxy analogues are more
 preferred and the 7-deaza-2'-deoxy guanosine (7-deaza-2'-dGTP) analogue is
 further preferred.
 In addition to using an analogue of guanosine, it is further preferred that
 the method of the present invention is performed in a reaction mixture
 that is substantially free of both GTP and dGTP.
 In the preferred embodiment, the polymerizations, or chain extension
 reaction, is performed in standard PCR buffer (50 mM Kcl, 10 mM Tris-Hcl,
 pH 8.3, 15 mM MgCl.sub.2, 0.001% (w/v) gelatin, (Saiki, Primer-Directed
 Enzymatic Amplification of DNA with a Thermostable DNA Polymerase, Science
 239: 487-91 (1988)) with the addition of 0.5-1 .mu.g denatured genomic
 DNA, 50 pmoles of each oligonucleotide primer, 2.5 units of Taq
 polymerase, and 10% DMSO. The reaction contained 320 .mu.M each of dATP,
 dCTP and dTTP, but was modified such that 320 .mu.m 7-deaza-2'-dGTP was
 used in place of dGTP.
 The following examples refer to the use of the invention to detect the
 presence of amplified GC-rich sequences in individuals afflicted with the
 fragile X syndrome, in male and female carriers of the pre-mutation for
 this condition, and in control individuals.
 The GTP analog used in these examples was 7-deaza-2'-dGTP. However, when
 7-deaza-2'-GTP was diluted with dGTP under the conditions employed in
 these examples, the higher molecular weight species were not detected
 (FIG. 1). Thus, it is preferred that the PCR reaction mixture is
 substantially free of GTP and dGTP.
 In alternative embodiments of the present invention, other constituents
 which improve the replication or transcription of GC rich nucleic acids
 such as DMSO and glycerol are employed.
 Conventional cloning and expression procedures can be adapted to employ the
 PCR method of the present invention. For example, in a conventional
 process for cloning one or more selected nucleic acids that are GC rich, a
 DNA can be amplified in quantity before cloning by using a PCR process
 that is substantially free of GTP and dGTP, but contains an analog of GTP
 or dGTP. Such a process might include: (i) adding a restriction enzyme to
 the product of the amplified nucleic acid in a manner effective to obtain
 cleaved products containing a selected DNA sequence; (ii) ligating such a
 cleaved product containing said selected DNA sequence in a manner
 effective to make a recombinant molecule; (iii) purifying, desalting
 and/or concentrating such cleaved products; (iv) sequencing said
 recombinant molecule containing the selected DNA sequence; (v) expressing
 the protein encoded by the specific nucleic acid sequence; and (vi)
 ligating such cleaved products into a new nucleic acid in a specific
 orientation.
 Several individuals afflicted with the fragile X syndrome and some of the
 members of their families were analyzed using our modified PCR assay. In
 some cases, genomic DNA was isolated from peripheral blood lymphocytes or
 cultured amniotic cells obtained from the afflicted individuals and their
 family members. Other samples were obtained directly from amniotic fluid
 without culture, or from crude cell lysates without DNA extraction.
 Methods for isolating genomic DNA are described in more detail in Kunkel,
 Analysis of Human Y Chromosome Specific Reiterated DNA in Chromosome
 Variants, Pro. Nat'l Acad. Sci. 74: 1245-49 (1977), which is hereby
 incorporated by reference.
 Oligodeoxyribonucleotide primers specific for a portion of the published
 FMR-I cDNA sequence (Verkerk, et al., 1991) were synthesized by cyanoethyl
 phosphoramidite Chemistry on a Biosearch/Milligen Model 8700 DNA
 Synthesizer and purified by HPLC. The sequence of the sense primer was 5'
 GACGGAGGCGCCCGTGCCAGG 3' (corresponding to nucleotides 1-21 of the FMR-I
 cDNA sequence) and that of the antisense primer was 5'
 TCCTCCATCTTCTCTTCAGCCCT 3' (corresponding to nucleotides 203-181 of the
 FMR-I cDNA sequence). Based on the published sequence (Verkerk, et al.
 1991) and our primer selection, the amplified product was predicted to be
 203 bp long and to contain a 90 bp CGG-rich region. The Verkerk reference
 is hereby incorporated by reference.
 The PCR amplifications were performed in standard PCR buffer (50 mM Kcl, 10
 mM Tris-Hcl, pH 8.3, 15 mM MgCl.sub.2, 0.001% (w/v) gelatin) with the
 addition of 0.5-1 ug denatured genomic DNA, 50 pmoles of each
 oligonucleotide primer, 2.5 units of Taq polymerase, and 10% DMSO. The
 reaction contained 320 .mu.M each of dATP, dCTP and TTP, but was modified
 such that 320 .mu.M 7-deaza-2'-dGTP was used in place of dGTP. Use of this
 modified nucleotide significantly increased the amount of specific PCR
 product generated with this set of primers and permitted amplification and
 detection of the very large alleles present in affected individuals, the
 detection of which alleles is not possible by the prior methods. The
 reactions were subjected to 40 cycles of denaturation at 97.degree. C. for
 30 sec, annealing at 55.degree. C. for 60 sec and elongation at 72.degree.
 C. for 60 sec.
 An aliquot of each reaction was analyzed by agarose gel electrophoresis.
 The PCR products could not be directly visualized by ethidium bromide
 staining, so Southern blot analysis was used to increase the visibility of
 these products. The DNA was transferred to a nylon membrane. The membrane
 was prehybridized at 42.degree. C. in a 0.9M sodium chloride, 0.09M sodium
 citrate solution ("6.times. SSC"), 5.times. Denhardt's solution, 0.5% SDS
 (sodium dodecyl sulfate) and 100 .mu.g/ml denatured carrier DNA. (See
 Manidatis et al.) Hybridizations were performed by the addition of
 oligodeoxyribonucleotide probes which had been radioactively labeled with
 gamma .sup.32 P-ATP using T4 polynucleotide kinase. After overnight
 hybridization, the filters were washed at room temperature for 15 min in
 6.times. SSC, 0.5% SDS, and then at 56.degree. C. for 30 min in 2.times.
 SSC, 05% SDS and subjected to autoradiography. One oligonucleotide probe
 (A) corresponded to nucleotides 127-151 of the FMR-I cDNA (5'
 CTGGGCCTCGAGCGCCCGCAGCCCA 3'); the other (B) was homologous to the CGG
 repeat region (5' [CGG].sub.5 3') which corresponds to nucleotides 37-126.
 EXAMPLE 1
 DNA isolated from: (1) a normal individual (lanes 1); (2) a fragile X
 carrier male (lanes 2); (3) a male afflicted with the fragile X syndrome
 (lanes 3); and (4) a female fragile X carrier (lanes 4) were subjected to
 PCR in the presence of different proportions of 7-deaza-2'-dGTP to dGTP
 (100:0; 75:25; 50:50). The PCR products were analyzed by blot
 hybridization using a probe B (described above) complementary to the CGG
 repeat region of the FMR-1 locus. FIG. 1 shows the results of this
 analysis. Note that the high molecular weight bands were detected only in
 the presence of 100% 7-deaza-2'-dGTP, 0% dGTP. In other words, the fully
 mutated fragile X gene was only detected when the PCR reaction mixture was
 substantially free of dGTP.
 EXAMPLE 2
 DNA samples were collected from fragile X family N43 and subjected to PCR,
 electrophoresis, and hybridization, as described above. The result of
 these analyses are illustrated in FIG. 2 (probe A results are on top and
 probe B on the bottom). Exposure times were 16 hours and 4 hours,
 respectively. Lane (-) is a no DNA control (primers only); lane (C) is a
 random control female DNA sample. DNA size markers, in bp, are indicated
 on the left. Only the 200 bp fragment was detected in DNAs from the
 non-carrier spouses: i.e., the afflicted individual's father and
 grandmother. A PCR product of approximately 400 bp was detected in the
 phenotypically normal carrier grandfather. The sequence was transmitted,
 with a small apparent increase in size, to his daughter. The fragile X
 positive grandson showed a PCR product of about 640 bp, indicating a
 significant increase in this region.
 Sequential hybridization with probes A (FIG. 2A) and B (FIG. 2B) yielded
 similar patterns. However, the altered sequences were more readily
 detected with the B probe. This suggests that the alterations involve an
 amplification of the CGG sequence, thus increasing the amount of PCR
 product sequence which is homologous to the CGG probe (probe B).
 EXAMPLE 3
 FIG. 3 illustrates the analysis of a second family (fragile X family N6)
 using the method of the present invention with only probe B. In this
 family, DNA markers had indicated that the grandfather was most likely a
 carrier and both of his daughters were known carriers. The analysis was
 performed as described in Example 2, except that only probe B was used.
 Exposure time was 3 hours. The carrier grandfather transmitted his
 amplified region, seen as a band of approximately 400 bp, to both of his
 carrier daughters. Both daughters show similar complex amplification
 patterns: in addition to the normal 200 bp band, both had bands of
 approximately 400, 530 and 650 bp.
 One daughter had an unaffected son with a normal band of 200 bp. A second
 pregnancy resulted in a cytogenetically positive male fetus which was
 terminated. DNA from this prenatal specimen lacked a normal band, but
 instead contained a heterogeneous smear extending from 400 to
 approximately 5000 bp.
 The other daughter had a cytogenetically positive, affected daughter with a
 200 bp band and a faint band of approximately 400 bp. Her affected son
 showed an amplified band of approximately 1000 bp. This son, his sister
 and parents have been previously studied by genomic Southern blot analysis
 using probe Ox1.9, and by cytogenetics (family 5; figure Sd; Nakahori et
 al., Molecular Heterogeneity of the Fragile X Syndrome, Nucl. Acids Res.
 19 4355-59, 1991). The affected son was of interest because he had been
 cytogenetically negative on several occasions. In the latter study, the
 carrier mother showed no abnormal DNA pattern, whereas our study clearly
 indicated the presence of an altered band. In our study the affected
 daughter also showed a pattern similar to the mother, although the altered
 allele was of lesser intensity.
 We have consistently observed similar results in studies of 38 affected
 males, 12 carrier males and 60 affected and unaffected carrier females,
 selected from 34 fragile X families. All affected males showed large bands
 and/or smears with probe B of up to 6 Kb in length.
 The results illustrate the potential for using our modified PCR to rapidly
 provide information about the presence and nature of the fragile X
 mutation. With this approach, it should be possible to quickly define
 alterations at the FMR-1 locus. Since all affected fragile X individuals
 appear to have a mother who is a carrier (Brown, 1990), it is now feasible
 to offer screening tests using a PCR-based method for detection of all
 carriers. The pregnancies of carriers can be monitored and the risk for
 the fragile X syndrome greatly reduced or eliminated.
 EXAMPLE 4
 A peripheral blood sample from an individual believed to have an
 Epstein-Barr virus infection is collected. Primers for the GC-rich
 terminal repeat region of the Epstein-Barr nucleic acid sequence are added
 along with other PCR reagents. The results are then compared to a standard
 to determine the clonality of infection in the individual who provided the
 sample.
 In summary, the present invention improves and extends the applicability of
 the PCR assay to GC-rich nucleic acid sequences. It makes possible for the
 first time the detection by the PCR method of high molecular weight
 species of GC-rich sequences present in the fragile X syndrome. The
 process is especially useful in detecting nucleic acid sequences that are
 initially present in only very small amounts and in detecting nucleotide
 variations using sequence-specific oligonucleotides. Also, the
 amplification process herein can be used for molecular cloning and
 sequencing. The process herein results in increased yields of amplified
 product, greater specificity, and fewer steps necessary to carry out the
 amplification procedure, over what has been previously disclosed. An
 advantage of the method of the present invention over the prior art is the
 ability to perform analysis of patient samples without necessitating the
 time and expense of tissue culture.
 Other modifications of the above-described embodiments of the invention
 that are obvious to those of skill in the area of molecular biology and
 related disciplines are intended to be within the scope of the following
 claims.