Abstract:
A process for detecting mutations in the gene responsible for galactosemia, galactose-1-phosphate uridyl transferase (GALT), is described. In one embodiment, the process can be used to detect over 85% of the mutations known to cause galactosemia in the United States population by using six different oligonucleotide probes, which span single-nucleotide Missense or nonsense mutations in the GALT gene. Hybridization conditions which can distinguish a single nucleotide mismatch are used to detect both the presence and zygosity of mutations in the GALT gene to aid in genetic counseling. A kit for use in detecting mutations in the GALT gene is also disclosed.

Description:
[0001]    This application claims priority in U.S. Provisional Application No. 60/103,286 filed Oct. 6, 1998. 
     
    
       [0002] The government may have certain rights in this invention, which was funded in part by National Institute of Child Health and Human Development Grant PO-1 HD 29847-01 and U.S. Public Health Services Grant M01-RR00039. 
     
    
     
       FIELD OF THE INVENTION  
         [0003]    The field of this invention is the diagnosis of galactosemia using nucleic acid-based techniques.  
         BACKGROUND OF THE INVENTION  
         [0004]    Galactosemia, an inherited metabolic disorder of milk sugar metabolism, is caused by certain mutations in the gene coding for the enzyme, galactose-1-phosphate uridyl transferase (GALT), which result in defective enzyme activity. This disorder is potentially fatal, so newborns throughout the U.S. and developed countries are screened for galactosemia, which when detected and treated early, saves lives and money. Evaluation of outcome in galactosemic patients began in the 1980s, and by 1990, an enigma was revealed (for example, see Kornrower, G M,  J. Inher. Metab. Dis.,  1982, 5:96-104; Waggoner and Buist, 1993 , Intenat. Pediatr.  8:97-100). Although neonatal infants were screened and treated within days of life with a galactose-restricted diet, older children had unexpectedly poor outcomes. Dysfunctions included ovarian failure, verbal dyspraxia, growth and developmental delays, and neurological signs of cortical and extrapyramidal tract impairment. Since the GALT enzyme structure is highly conserved throughout evolution, the human GALT cDNA was cloned using probes homologous to known sequences in the bacterial enzyme. When the human cDNA and gene were sequenced (see Flach, Reichardt, and Elsas, 1990, Mol. Biol. Med. 7:365-369; and Leslie et al. 1992, Genomics 14:474-480), the first mutations in the GALT gene were associated with human galactosemia (Reichardt et al. 1991, Am. J. Hum. Genet. 49:860-867; Reichardt et al, 1992, Genomics 12: 596-600) and relationships between the mutations and the resulting phenotype were being reported (Elsas et al. 1993, Internat Pediatr. 8:101). The Q188R mutation, first reported in 1991, is the most common mutation among galactosemic patients of Caucasian ethnicity (Reichert et al. 1991, Ibid.) and causes a complete loss in GALT activity. The S135L mutation is common among African-Americans (Lai et al., J. Pediatr 1996, 128: 89-95), and results in severe reduction in GALT activity. The N314D mutation, also known as the Duarte allele (Elsas et al., 1994, Am J. Hum Genet 54: 1030-1036), causes an instability in the GALT protein that leads to a 50% reduction in activity in homozygous patients. The K285N mutation is the second most common mutation among white galactosemic patients, and is the most prevalent among patients in southern Germany and Austria (Leslie et al., 1992, Ibid.). Other common mutations identified in the white population are Y209C and L195P (Reichart et al. 1992, Ibid.).  
           [0005]    These reports led to the realizations that different GALT mutations can result in different phenotypes and that therapy recommendations may vary depending on the specific mutation.  
           [0006]    Methods currently used in newborn screening determine only the level of enzymatic activity or the accumulation of precursors. Results from current techniques are altered by the sampling parameters, such as ambient temperature and time of feeding relative to collection of sample. Also, they do not reveal the specific mutations that cause the change in enzyme activity. Certain mutations cause a mild disease, while others cause severe effects. Identifying the mutation is necessary to make a diagnosis, initiate appropriate therapy, estimate prognosis, and provide appropriate genetic counseling for the family.  
           [0007]    There remains a need for a test with increased sensitivity and specificity for screening and diagnosis of galactosemia and asymptomatic carriers of galactosemia. There is a need for a test that can be run simultaneously on a large number of samples, such as in a micro titer plate format now commonly in use in clinical diagnostic laboratories. There is a need for a testing protocol that can quickly and specifically identify the majority of the alleles that can contribute to galactosemia in the human populations.  
           [0008]    The claimed invention describes a nucleic acid-based method that can detect and specify the mutation involved in over 85% of individuals with some form of galactosemia rapidly and economically. Such a screen would be applicable to universal newborn screening and should supplement or completely replace current screening strategies. The screen can also be used to detect heterozygous mutations and the presence of multiple mutations in the GALT gene, which are necessary for understanding and counseling heritability and recurrence risks to family members.  
         SUMMARY OF INVENTION  
         [0009]    The invention as claimed is a nucleic acid-based method that can detect and specify the mutation involved in the great majority of individuals with some form of galactosemia rapidly and economically. The method involves the detection of the mutation in amplified DNA from a patient utilizing probes that span the mutation. Six mutations are identified as being responsible for over 85% of the mutations in the United States population, and a detection kit is described for detecting these six mutations. The methods of this invention can also be used to determine the zygosity of the mutations and to detect the presence of multiple mutations in the GALT gene, both of which are necessary for understanding and counseling heritability and recurrence risks to family members.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0010]    Definitions:  
           [0011]    “GALT” means the enzyme galactose-1-phosphate uridyltransferase.  
           [0012]    “Galactosemia” is a deficiency in the level or expression of GALT which results in detectable deviations from normalcy in animals.  
           [0013]    As used in the claims, “a” can mean one or more, depending on the context of the claim.  
           [0014]    Detailed Description of Invention  
           [0015]    The invention described herein involves the use of a group or set of DNA probes to detect mutations in the GALT gene. Prior to this invention, only enzyme activity analyses have been used in clinical screening applications. With the practice of this invention, a rapid and economical screening system for detecting galactosemia in human populations, particularly in newborns, is provided. Based on the discovery of the frequency of the K285N mutation and the existence and frequency of the Y209C mutation, a process for detecting specific mutations in the GALT gene that cause over 85% of the mutations leading to galactosemia currently known in the U.S. population is provided. A preferred embodiment of this invention comprises assaying for the presence of six mutations, listed in Table 1A. In a survey of newborns the prevalence of these mutations is shown in Table 1B. If desired, this process can be followed by additional screening for less common mutations, e.g. listed in Table 2. The process comprises the collection of an RNA- or DNA-containing sample from a patient or a newborn infant, amplification of the DNA (following reverse transcription of the RNA, if needed) that encodes either a portion of or the entire GALT gene, hybridization of the amplified DNA, either serially or in parallel, with each member of a set of detectable probes, each probe being at least 10 nucleotides in length, the set comprising probes having sequences complementary to the sequences set forth in Table 1A, under hybridization conditions that allow only exactly complementary strands to remain as hybrids, and detecting the presence or absence of the hybrids.  
                       TABLE 1A                               Approx % found in       Designation #   Sequence*   galactosemic population                   S135L   TGGTTGGAT   62% of African-American population       Q188R   TGCCGGGTA   70% of white population       L195P   TTCCCGCCA    8% of all ethnic groups       K285N   CCAATTATG   17% of white populations       Y209C   GCCTGTAAG    6-20% of all ethnic groups       N314D   CTGGGACCA    8% of all ethnic groups       [deletion]   [absence of signal   in Ashkenazi Jewish population           with any probe]                                  
 
           [0016]    [0016]                                 TABLE 1B                           Observed Prevalence In 250 Patients (500 Mutant Alleles)                Designation   # of Alleles   Percent of Total                       Q188R   270   54.0           S135L    42    8.4           K285N    24    4.8           L195P    8    1.6           Y209C    6    1.2           F171S    5    1.0           [deletion]    5    1.0           Private    90   18.0           Unknown    50   10.0               500   100.00                        
           [0017]    [0017]                                                                             TABLE 2                           A. Missense/Nonsense mutations                Designation #   Sequence*   Designation #   Sequence*                       D28Y   TAC   Q212H   CCG           I32N   AAC   L217P n     CCA           Q38P   CCG   L226P n     CCA           V44L   TTG   R231H   CAT           V44M   ATG   W249R   AGG           R51L   CTC   Y251C   TGC           G55C   TGT   Y251S n     TCC           L62M   ATG   R258C n     TGT           R67C   TGC   R259W   TGG           L74P   CCG   R262P n     CCG           A81T   ACC   R273G   GGT           N97S   AGC   L282V n     GTC           D98N   AAC   L289R n     CGC           D113N n     AAT   E291K   AAG           H114L n     CTT   E308K   AAG           F117S n     TCC   Q317R   CGG           Q118H n     CAC   Q317H n     CAT           R123G   GGA   H319Q   CAA           R123Q n     CAA   A320T   ACT           V125A n     GCC   Y323H   CAC           K127E n     GAG   Y323D   GAC           C130Y n     TAC   P324S   TCT           H132Y n     TAC   P325L n     CTG           T138M   ATG   R328H n     CAC           L139P   CCG   S329F   TTT           M142V   GTG   A330V   GTG           M142K   AAG   R333W   TGG           S143L   TTG   R333G   GGG           R148W   TGG   R333Q   CAG           R148Q   CAG   K334R   AGA           R148G n     GGG   M336L n     TTG           V150L   CTT   Q344K n     AAG           V151A   GCT   T350A   GCC           W154G n     GGG   Q54Stop n     TAG           F171S   TCT   R80Stop   TGA           G179D   GAC   W154Stop n     TGA           P183T   ACC   R204Stop   TGA           H184Q n     CAA   Q212Stop   TAG           S192N   AAG   W249Stop   TGA           F194L   CTG   L264Stop n     TAG           I198M n     ATG   W316Stop   TAG           I198T n     ACT   E340Stop   TAA           A199T   ACC   Q353Stop   TAG           R201H   CAT   Y366Stop   TAA           E203K   AAG   Q370Stop   TAG           Y209S   TCT                        B. Small Insertions/Deletions                        528insG   insG at base 528           S112fs insA   insA at base 333           W134fs delT   del T at base 400           ΔC (bp1677) n     ΔC (bp1677)           L256/P257fs   at bases 768-770,               delete GCC-fs           Δamino acids n     Δamino acids           #260-263   #260-263           ΔT (bp2359)Th   ΔT (bp2359)           ΔC (bp2756) n     ΔC (bp2756)           ΔC (bp2782)   ΔC (bp2782)           L327delC   base 979, del C-fs           P351fsdelC   base 1051, del C-fs                        1. Splice Site Mutations                        318A→G   base 318A→G           TVSC   base 956A→C           IVS3nt + 29G→C   c.328 + 29 G→C           IVS4nt + 1   c.377 + G→C           IVS4nt − 27G→C   c.378 − 27 G→C           IVS5nt + 62G→A   c.507 + 62 G→A           IVSFnt1 n     base 1472 G→A           IVSF   base 1631 A→G           IVS7nt + 2   c.687 + 2 T→C           IVS8nt + 13   c.820 + 13 A→G           IVS8nt + 32   c.820 + 32 A→G           IVS8nt + 58   c.820 + 58 G→T           IVSHnt13   base 2207 A→G           IVSJ   C→G                                                            
           [0018]    The invention herein described provides the novel mutation, Y209C, in which the amino acid tyrosine is replaced with cysteine at amino acid 209. When a gene carrying the Y209C mutation is analyzed for expression in  E. coli , it is scored as a “null” mutation, i.e. there is no significant GALT activity detected. In one embodiment, the invention comprises a process for detecting this mutation in the GALT gene comprising amplifying the GALT gene, or the portion containing exon VII, hybridizing the amplified DNA with a probe at least 10 nucleotides in length that comprises sequence complementary to GCCTGTAAG, and detecting the presence or absence of this hybrid. In one embodiment, the invention comprises a set of probes comprising a probe for the Y209C mutation, as described above, and a probe for at least one other mutation chosen from the groups of mutations listed in Table 1A and Table 2. In another embodiment, the invention provides a set of probes comprising at least one of the novel mutations listed in Table 2 (i.e. those marked with the superscript “ n ” and at least one other mutation chosen from the groups of mutations listed in Table 1A and Table 2.  
           [0019]    The DNA hybrids produced by the processes of this invention can be detected by any of a number of methods known in the art, e.g. radioactive, chemiluminescent, fluorescent or bioluminescent labeling of the probe, antibody-based detection of a ligand attached to the probe, or detection of double-stranded nucleic acid. Specific examples of labels are digoxigenin, fluorescein, luciferases, and aequorin. Alternatively, the probe can be labeled with biotin and detected with avidin or streptavidin conjugates.  
           [0020]    This process can be used to detect carriers of galactosemia, who may themselves be asymptomatic or mildly symptomatic and therefore undiagnosed. Carriers are heterozygous for a mutation, for example one of those listed in Table 1A. Carriers have not previously been identified by the existing screening methods, since they do not demonstrate a significant change in GALT activity.  
           [0021]    In a specific embodiment, a portion of the GALT gene comprising exons 5-10 (e.g. nucleotides 1153-2863, GenBank Accession No. M96264; SEQ ID NO: 7) is amplified from the subject&#39;s DNA. Alternatively, the GALT gene can be amplified in sections, and the resulting amplicons can be hybridized only with those oligonucleotides carrying a mutation in the amplified region of the gene.  
           [0022]    The process of this invention can be used to determine the zygosity of a mutation by hybridizing the amplified DNA to one or more probes comprising the wild-type sequences that correspond, for example, to the mutated sequences listed in Table 1A.  
           [0023]    In specific embodiments, the probes used to detect the GALT mutations can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.  
           [0024]    In a specific embodiment, the probes used to detect the set of mutations, such as the set in Table 1A, are all of the same length, between 10 and 25 nucleotides, and the mutated nucleotide (or the wild-type nucleotide corresponding in position to the location of the mutation) is positioned exactly in the middle of each probe, in the case of probes with an odd number of nucleotides, or, in the case of a probe with an even number of nucleotides, n, the mutation would be located at n/2±1. In such an embodiment, identical hybridization conditions can be used for each probe.  
           [0025]    Alternatively, the mutation can be positioned anywhere in the probe that is at least 3 nucleotides from either end of the probe. In using such an alternative approach for detecting a set of mutations, the mutated nucleotide should be in the same position in each probe.  
           [0026]    In an alternative embodiment, allele-specific amplification is employed resulting in the amplification of only selected GALT gene alleles. In this embodiment, probes are designed such that the 3′ end of one primer is the nucleotide which is mutated (see Blasczyk R, J Wehling et al. 1997 Allele-specific PCR amplification of factor V Leiden to identify patients at risk for thromboembolism,  Beitr Infusionsther Transfusionsmed  34: 236-41).  
           [0027]    In a specific embodiment, tetramethyl ammonium chloride (TMAC) is included in the hybridization buffer, and the reaction is carried out at 55 C. Typically, the temperature of the hybridization is used to discriminate between an exact sequence match and a mismatch, and the temperature at which a given hybrid will “melt”, i.e. dissociate into the two complementary single strands, is determined by the length and nucleotide composition of the strands. To minimize the variation in the reaction temperature required to discriminate between the desired exact-match hybrid and the mismatched hybrid, trimethyl ammonium chloride (TMAC) is added to the hybridization buffer. TMAC greatly minimizes the effect of nucleotide composition on the hybridization reaction, so that length of the oligonucleotide becomes the determining factor, which can be easily controlled. Other salts can alternatively be used with similar effects as TMAC (see, for example, Mechior, Jr., W B and P H Von Hippel, 1973, Proc. Natl. Acad. Sci (USA) 70(2): 298-302). In a specific embodiment, probes of 21 nucleotides, with the mutation in the exact middle of the probe, as set forth herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, are used in the process.  
           [0028]    In another embodiment, a kit is provided for use, for example, by diagnostic laboratories in hospitals, clinics or research centers that contains the necessary components to perform a rapid and economical screen for the six most common mutations in the GALT gene. Such a kit comprises a set of probes comprising sequences complementary to the sequences set forth in Table 1A, and a set of amplification primers, complementary to sequences selected from the known sequence of the human GALT gene (e.g. GenBank Accession No. M96264 or SEQ ID NO:7) to amplify the desired portion of or the entire GALT gene in a patient sample. In a specific embodiment, the primers comprise a single pair which amplify a region of the human GALT gene that comprises at least nucleotides 1153-2748 (numbering as in the above-referenced GenBank accession, SEQ ID NO: 8). Amplification primers can be 14 to 30 nucleotides in length, although longer ones can also be used.  
           [0029]    In another embodiment, the detection method used is an ELISA-based oligonucleotide ligation assay. Probes spanning the various GALT mutations described herein can be divided into a pair of smaller probes or, alternatively, new probe pairs can be designed so that their ends meet within a few base pairs of the mutation (or corresponding wild-type sequence). Such a pair of probes, spanning the sequence of a GALT mutation, are hybridized to the patient&#39;s amplified GALT DNA, then ligated, and the probes are then analyzed to determine the presence of a single large probe, which would be the product of the ligation of the perfectly matched pair of smaller probes (see Nickerson, D A, R Kaiser, S. Lappin, J. Stewart, L. Hood, and U. Landegren, 1990, Automated DNA diagnostics using ELISA-based oligonucleotide ligation assay; Proc. Natl. Acad. Sci. USA 87:8923-8927). The absence of a large product indicates the absence of the GALT mutation represented by the pair of probes.  
           [0030]    In another embodiment involving a solution-based assay, detection of the GALT gene alleles involves the use of two different labels. For example, the DNA amplification is performed with primers that are labeled with digoxigenin. Multiple alleles can be analyzed by including primers labeled with a different detectable moiety for each allele. The amplified product is melted in solution, and allele-specific probes (as described herein) conjugated with biotin are added to the solution and hybrids are allowed to form. The solution containing these hybrids is added to an ELISA plate coated with avidin or streptavidin to capture the allele-specific hybrids. The hybrids are detected through the presence of digoxigenin.  
           [0031]    Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more filly describe the state of the art to which this invention pertains. 
       
    
    
     EXAMPLES  
     Example 1  
       [0032]    A. Oligonucleotides Useful in Detecting GALT Gene Mutations  
         [0033]    Oligonucleotides useful in detecting the GALT gene mutation can be synthesized by techniques known in the art. The oligonucleotides can be from nine to twenty-five nucleotides in length; nucleotides shorter than 9 nucleotides will not provide accurate and reproducible results. Nucleotides longer than 25 nucleotides in length for detecting a single mutation in GALT can be used, but they provide no advantages over oligonucleotides 9-25 nucleotides in length. The single nucleotide mutation can be located anywhere in the oligonucleotide, as long as it is at least 3 nucleotides from either end of the oligonucleotide. One set of oligonucleotides of this invention, wherein the mutation is located exactly in the middle of the oligonucleotide, is presented in Table 3.  
         [0034]    B. Amplification of DNA  
         [0035]    1. GALT Gene PCR  
         [0036]    Samples are collected from any tissue or organ of a patient from which RNA or DNA can be amplified. For example, DNA in blood collected and dried onto filter paper can be amplified. Alternatively, a buffy coat fraction from freshly collected blood is preferred for RNA amplification. Patient&#39;s DNA or RNA can be amplified in three sections using the following primers:  
                                       Primer Name   Nucleotide Sequence   SEQ ID NO:                   F-S135L   TGT AGT GGC TCT AGC TCT GG   10       R-S135L   TAT CAG ATA GGG ATA CGG AGC   11       F-Q188R   GTC AGG AGG GAG TTG ACT TGG   12       R-Q1S8R   GCC CTT TTA CAG TTA TCC AGG   13       F-N314D   TCA AGC AGG GGA TCC TGG GAG   14       R-N314D   ATG TCT GAA GGT TCA CAC TCC   15                  
 
         [0037]    2. GALT Allele-specific Amplification  
         [0038]    The presence of a specific GALT mutation or wild type can be detected by using allele specific amplification. A synthetic oligonucleotide, designed to end with a nucleotide that creates the mutation, is used as one of the primers in polymerase chain reaction amplification (for example, see Blasczyk R, J. Wehling et al. 1997 Allele-specific PCR amplification of factor V Leiden to identify patients at risk for thromboembolism,  Beitr Infusionsther Transfusionsmed  34:23641). One or more adjacent nucleotides of the primer may also be modified to increase the discrimination between allele-specific and non-specific hybridization. In addition, this technique can also be used to simultaneously detect multiple mutations. Primers for each of the different GALT alleles are designed to result in differing lengths of the PCR product, or different detectable moieties (e.g. fluorescent molecules, biotin, digoxigenin, or antibody ligands) are attached to the allele-specific oligonucleotide primers.  
         [0039]    For example, the following primer sets can be used to detect two specific mutations of this invention. For the mutation S135L, the following primers can be used:  
         [0040]    Mutated allele primer: 5′ GGT CAT GTG CTT CCA CCC CTG GTT 3′ 
         [0041]    Wild-type allele primer: 5′ GGT CAT GTG CTT CCA CCC CTG GTC 3′ 
         [0042]    Common reverse primer: 5′ CTG CAC CCA AGG GTA CTG GGC ACC 3′ 
         [0043]    PCR Product=127 base pairs  
         [0044]    For mutation Q188R, the following primers can be used:  
         [0045]    Mutated allele primer: 5′ TTC TAA CCC CCA CCC CCA CTG CCG 3′ 
         [0046]    Wild-type allele primer: 5′ TTC TAA CCC CCA CCC CCA CTG CCA 3′ 
         [0047]    Common reverse primer: 5′ CAA AAG CAG AGA AGA ACA GGC AGG 3′ 
         [0048]    PCR Product=176 base pairs  
         [0049]    The common primer is used in combination with either one of the other primers, the 3′ end of which is the specific nucleotide that is different in the mutated allele. The GALT gene DNA from an individual with only the wild-type form of the gene will only produce the appropriately sized product with the wild-type primer and none with the mutated allele primer. A heterozygous individual will produce the appropriately sized product with both primers, and a homozygous individual will not produce any product with the wild-type primer but will yield the appropriately sized product with the mutated allele primer.  
         [0050]    C. Hybridization Techniques  
         [0051]    When two DNA strands associate through base-pairing, it is referred to as hybridization. Hybridization is strongest when the two strands are exactly complementary. The stability of a hybrid is also a function of temperature, the ionic concentration of the medium, the length of the two strands, and the nucleotide composition, i.e. %A-T or %G-C. Under identical conditions, two strands that are not exactly complementary, differing by even one nucleotide, will be less stable and will disassociate at a temperature at which exactly complementary hybrids remain paired.  
         [0052]    This property of mismatched hybrids provides a method to detect mutations in the GALT gene. The GALT sequence in the DNA of the subject, e.g. a newborn or a patient needing a diagnosis, is amplified, using techniques known in the art, such as polymerase chain reaction or cDNA synthesis, to produce copies of the gene which are then immobilized on a support, such as a nylon membrane (see Sambrook, et al.). The DNA on the support is incubated with a solution containing a labeled oligonucleotide from Example 1 at a temperature that promotes formation of hybrids. The support is then washed at a temperature at which only exact hybrids would be stable. The presence of the label is then measured; if the subject&#39;s DNA had the mutation corresponding to the oligonucleotide used in the assay, the label would be detected on the support.  
         [0053]    Many subjects&#39; DNA can be tested in a microtiter plate format. Additionally, a pool of probes, containing all or a subset of the mutations listed in Table 1A or 1B, can be used simultaneously to make an initial qualitative determination of whether the subject&#39;s DNA contains any mutations in the GALT gene. Parallel hybridizations using oligonucleotides for the normal sequence corresponding to the mutated sequence can be performed to determine zygosity. It is also possible to put a unique and distinguishable label on each probe, so that one could determine the presence of different mutations in a single hybridization mixture.  
       Example 2  
       [0054]    GALT Mismatched Hybrids Assay  
         [0055]    To use multiple probes in a single hybridization format, an assay was developed by determining an optimal length for the probes so that the same temperature changes would result in the melting of the mismatched hybrids. Oligonucleotides between 9 and 25 nucleotides could satisfy this requirement, and those of 21 nucleotides, with the mutation at position 11, (see table 3) were found to be especially well-suited to a single temperature for disassociation of the hybrid. In addition, the use of tetramethyl ammonium chloride (TMAC) to the hybridization buffer allowed the hybridization temperature to be standardized at 55 C for all probes.  
         [0056]    Hybridization Buffers and Techniques.  
         [0057]    Amplified DNA in 5 μl aliquots are spotted on a prewetted positively charged nylon membrane, denatured (using 0.5M NaOH, 2.0M NaCl, 25 mM EDTA) and UV-crosslinked. The membranes are prehybridized at 55° C. for 30 minutes in a hybridization buffer (3.0M TMAC, 0.6% SDS, 1.0 mM EDTA, 10 mM Na 3 PO 4 , pH 6.8, 5× Denhardt&#39;s solution, 40 μg/ml yeast RNA) followed by overnight hybridization in the same buffer and the same temperature containing 1-2×10 6  cpm/ml  32 P-end labeled oligonucleotides. Unlabeled oligonucleotides corresponding to the normal sequence are used at 10× concentration of the labeled mutant sequence in the hybridization. Similarly, in detecting the normal alleles, unlabeled oligonucleotides corresponding to the mutant sequence are used at 10× concentration of the normal sequence in the hybridization. Filters are then rinsed one time for 20 minutes at room temperature with wash buffer (3.0M TMAC, 0.6% SDS, 1.0 mM Na 3 PO 4 , pH 6.8), then washed again in wash buffer at 55° C. for 30 minutes. The membranes are sealed in plastic and set for autoradiography overnight. Table 3.  
                       TABLE 3                       Mutation   Sequence   Sequence ID NO:                   Y209C   CAGCAGGCCTGTAAGAGTCAG   1       S135L   CACCCCTGGTTGGATGTAACG   2       Q188R   CCCCACTGCCGGGTAAGGGTG   3       L195P   AGCAGTTTCCCGCCAGATATT   4       K285N   TCTTGACCAATTATGACAACC   5       N314D   GGCCAACTGGGACCATTGGCA   6                  
 
       Example 3  
       [0058]    Screening of a Galactosemia-Positive Patient for Mutation Type(s)  
         [0059]    It is sometimes desirable to determine the exact nature of the mutation or mutations that are contributing to a patient&#39;s galactosemia. Such a determination will become critical as gene therapy techniques are implemented to correct these deficiencies. One example for analyzing a single patient&#39;s GALT gene for the presence of any or all of the known mutations in the GALT gene is provided herein. Synthetic oligonucleotides carrying each of the mutations to be screened are immobilized individually onto a support, such as a membrane, for example, as “dots” on a nitrocellulose membrane. A sample of the patient&#39;s labeled, amplified GALT gene (see Example 1 above for methods of amplification) is hybridized to all the dots on the membrane under conditions that promote only the hybridization of exactly matched sequences. The membrane is washed to remove mismatched and nonspecific hybridizing material, and the membrane is exposed to the appropriate detection system for the label, e.g. film, in the case of a radioactive label, to reveal which synthetic oligonucleotides are hybridizing to the patients&#39; DNA.  
         [0060]    Alternatively, detection of a GALT gene mutation can be accomplished in solution, without the need for an immobilization support. An example of a solution-based analysis is the detection of a GALT gene mutation or a wild-type GALT gene by determining the melting temperature of the hybrid of the amplified DNA and the specific oligonucleotide (Ririe, K M, Rasmussen et al. 1997 Product differentiation by analysis of DNA melting curves during the polymerase chain reaction,  Anal Biochem  245(2): 154-60; Wittwer, C T, M G Herrmann et al. 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification,  Biotechniques  22(1): 130-1, 134-8). The melting temperature (Tm) of a hybrid is determined empirically by monitoring the absorbance of the solution as the temperature of the solution is raised incrementally. The Tm is typically defined as the temperature at which half of a single hybrid population is melted. When several different hybrids are present in the solution, there will be several different Tm values, representing the melting of each hybrid. The particular Tm value is determined by the G+C content of the hybrids, their length, and the ionic concentration of the solution. Mismatched hybrids will denature at a lower temperature than exactly matched hybrids. One or more of the probes can be hybridized to the amplified DNA and the melting curves of hybrids can be determined against small increments in temperature. Discrimination between multiple mutations can be achieved by using oligonucleotides of differing lengths or by attaching different detectable moieties, such as fluorescent, chemiluminescent, bioluminescent, or radioactive molecules, biotin, or antibody ligands, to the oligonucleotides.  
         [0061]    A specific application of this approach, known as fluorescence energy transfer, can be performed using a Lightcycler®, a device manufactured by Roche Diagnostics Ltd., a division of Roche Molecular Biochemicals, Mannheim, Germany (see Wittwer, C T, K M Ririe et al. 1997 The LightCycler®: a microvolume multisample fluorimeter with rapid temperature control.  Biotechniques  22(1): 176-81). For example, to detect mutation Q188R, the following primers are synthesized:  
         [0062]    Amplification Forward Primer: 5′ CTT TTG GCT TAA CAG AGC TCC G 3′ 
         [0063]    Amplification Reverse Primer: 5′TTC CCA TGT CCA CAG TGC TGG 3′ 
         [0064]    Anchor probe: 5′ GTC CCC GAG GTC ACC CAA AGA ACC G 3′ 
         [0065]    Detection wild-type probe: 5′ GGT GAC GGt CCA TTC CCA CA 3′ 
         [0066]    Detection mutated allele probe: 5′ GGT GAC GGc CCA TTC CCA CA 3′ 
         [0067]    (The lower case letter represents the nucleotide that is different between the mutated and wild-type alleles). The anchor probe forms one half of the detection probe set and carries a fluorescence quencher. The other half of the probe set has the allele-specific sequence. When the allele specific probe and the anchor probe are hybridized the fluorescence is quenched. Then, during melting at the Tm for the allele specific probe the hybrid melts and fluorescence is observed. In this example, the Tm for the hybrid formed with the wild-type probe is 56 C, while the Tm of the hybrid formed with the mutated allele probe is 65C.  
       Example 4  
       [0068]    ELISA-like Detection of Hybridization  
         [0069]    The hybridization of the DNA oligonucleotide probe to the amplified GALT DNA sequences can also be detected using an enzyme-linked immunodetection assay, which can be generally called an ELISA. The amplified DNA is labeled during the amplification reaction with digoxigenin, then hybridized with biotin-labeled probes. Under the hybridization conditions described above, a hybrid of the biotin-labeled probe and the digoxigenin-labeled DNA forms. These hybrids are introduced into avidin-coated microtiter plates, and the biotin-labeled probe is adsorbed to the plate. Digoxigenin-labeled DNA that is hybridized to the bound biotinylated probe is detected using an enzyme-coupled anti-digoxigenin antibodies. Detection signals other than an enzyme can be used to detect the anti-digoxigenin antibodies, as will be recognized by those skilled in the art. Examples of other detection signals include bioluminescent proteins, chemiluminescent or fluorescent labels, or radioactive labels.  
         [0070]    Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention. The texts of the references herein cited are hereby incorporated in their entirety by reference.