Method for simultaneously detecting multiple mutations in a DNA sample
This is a continuation of application Ser. No. 07/957,205, filed Oct. 6, 1992 now abandoned. 
    
    
     BACKGROUND 
     The ability to detect differences in DNA sequence (i.e. mutations) is of great importance in the field of medical genetics. For example, the detection of mutations directly in genomic DNA is essential for identifying polymorphisms for genetic studies, to determine the molecular basis of inherited diseases and to provide carrier and prenatal diagnosis for genetic counselling. Traditionally, detection of DNA variation has been performed by analysis of RFLPs using the Southern blotting technique (Southern, EM J Mol Biol 98:503-517 (1975)): Kan Y and A Dozy Nature 313:369-374 (1978); Wyman, A. and R. White Proc Natl. Acad. Sci. USA 77:6754-6758 (1980)). However, as this approach is relatively slow and technically demanding, new methods based on the polymerase chain reaction have been developed. These include (RFLP) analysis (Chehab et. al. Nature 329:293-294 (1987), the creation of artificial RFLPs by the use of primer-specified restriction-map modification (Hallassos A. et. al. Nucleic Acids Res. 17:3606 (1989)), hybridization to allele-specific oligonucleotides (ASOs) (Saiki et. al. Nature 324:163-166 (1986)) or detection of small deletions by determination of the size of the PCR product (Rommens et. al. Am J. Hum Genet 46:395-396 (1990)). Of these methods, only the ASO approach can be used to detect any point mutation or small deletion, as the other methods are all dependent on the nature of the mutation and the surrounding DNA sequence. 
     It is now becoming clear that, for many genetic diseases, there is more than one mutation responsible for the condition. For example, to date more than 225 cystic fibrosis (CF) disease causing mutations have been reported (CF Genetic Analysis Consortium, unpublished data), while not accounting for all cases of CF. Furthermore, the mutations can be closely spaced often within a few base pairs of each other. Examples of multimutational diseases include CF (Cutting G. et. al. Nature 346:366-369 (1990)); β-thalassaemia (Old JM et. al. Lancet 336:834-837 (1990)) Tay-Sachs disease (Myerowitz R. Proc Natl Acad Sci USA 85:3955-3959 (1988)) and Sickle cell anemia (Saiki, R. K. et. al. Science 230, 1350-1354 (1985)). The presence of multiple potential mutations makes the detection of these diseases complex. 
     A method which enables the simultaneous analysis of a sample for the presence of multiple mutations would be useful. 
     SUMMARY OF THE INVENTION 
     In general, the invention relates to a process for analyzing a DNA sample for the presence of multiple mutations simultaneously using allale specific oligonucleotide probes (ASOs). 
     According to the process, a DNA sample is hybridized with multiple mutation specific ASO probes of approximately the same length in the presence of an agent that eliminates disparities in ASO melting temperatures under stringent hybridization conditions. After an appropriate period of time, the sample can be washed to remove unhybridized ASO probes and the presence or absence of hybridization detected. The detection of hybridization being indicative of the presence of at least one mutation in the DNA sample. 
     In a preferred embodiment, the agent that eliminates disparities in ASO melting temperatures is a quaternary ammonium salt such as tetramethyl ammonium chloride (TMAC), which is included in the hybridization buffer. In a further preferred embodiment, the hybridization buffer additionally includes unlabelled allale specific oligonucleotides or fragments thereof which are complimentary to the normal, wild-type allale specific sequences. 
     The method described herein provides a rapid, cost-effective means to screen large numbers of samples simultaneously for multiple mutations including point mutations and small deletions at a particular genetic disease locus at one time, as well as multiple mutations in different genes located on the same or on different chromosomes. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is predicated on the surprising finding that in the presence of an agent that eliminates disparities in melting temperatures, multiple allele specific oligonucleotide (ASO) probes recognizing multiple regions on the same gene or multiple genes on the same or different chromosomes and having varying GC base-pair contents can be simultaneously hybridized under stringent conditions to a sample DNA. This result is surprising, since previously, for each ASO probe used in an assay, a separate hybridization reaction and wash procedure was required. 
     Based on this finding, the invention features, in general, a process for simultaneously detecting the presence or absence of multiple mutations (i.e. more than one mutation) in a DNA sample by hybridizing the sample with multiple allele specific oligonucleotide probes of approximately the same length under stringent conditions in the presence of an appropriate concentration of an agent that eliminates disparities in melting temperature and detecting hybridization as an indication of the presence of at least one mutation in the sample. 
     As used herein, the term &#34;mutation&#34; is meant to refer to a change in the base sequence of DNA from the normal, wild-type sequence. The most common mutations are substitutions, additions (i.e. insertions); rearrangements and deletions of one or more base. The term &#34;point mutation&#34; is generally used to denote a change in a single base pair. 
     For use in the invention, a DNA sample may be obtained from any cell source using methods which are well known in the art. For example, DNA can be obtained from: (i) blood leukocytes obtained from whole blood (e.g. via centrifugation); (ii) buccal cells obtained using a swab or cytobrush as described in Example 1 herein or using a mouthwash technique (Lench, N. et. al. The Lancet 1:1356-8 (Jun. 18, 1988,)); (iii) cervicovaginal cells obtained using a brush, swab or lavage (Burk, R. D. and C. Spitzer Am J Obstet Gynecol. 162:652-4 (1990); (iv) epithelial cells obtained from urine (Gasparini, P. et. al. N. Engl. J. Med 320:809 (1989) or hair roots (Higuchi, R. et. al. Nature 332:543-6 (1988); (v) fetal cells obtained from amniotic fluid, cord blood, chorionic villus tissue, cervical secretions or maternal blood (Bianchi, D. W. et. al Proc. Natl. Acad. Sci. USA 87:3279-83 (1990); and (vi) embryonic cells obtained from biopsied embryos. 
     Once obtained, a DNA sample can be prepared for hybridization using techniques which are well-known to one of skill in the art. For example, in order to increase the amount of DNA available for hybridization and thereby the signal obtained, amplification procedures, such as the polymerase chain reaction (PCR) can be employed (Saiki, R. K. et. al. Science 239:487-491 (1988)). 
     ASOs to be used in the subject invention can be designed to identify allele specific mutations of a gene. Preferably ASOs are synthesized in appropriate amounts, for example using a DNA synthesizer. ASOs can then be labelled with a detectable marker to generate ASO probes according to procedures which are well known in the art. Traditionally, ASOs have been radioactively end-labelled (e.g. using  32  P or  35  S). However, ASOs can also be labelled by non-isotopic methods (e.g. via direct or indirect attachment of fluorochromes or enzymes, or by various chemical modifications of the nucleic acid fragments that render them detectable immunochemically or by other affinity reactions). 
     ASOs which are to be used in a pool to detect multiple mutations in the same hybridization reaction according to the method of the subject invention, should all be approximately of the same length (i.e. approximately the same number of base pairs). The appropriate concentration of a particular ASO to be used in a pool can be determined empirically without requiring undue experimentation. For example, the optimal concentrations of each ASO used in a pool to probe the cystic fibrosis transmembrane regulator gene, as set forth in the following Example 1, was initially tested at a concentration of 0.03 pmol. If the signal produced was too light, the concentration was doubled until the appropriate signal intensity was obtained. It is important to point out that if the concentration of ASO used is too high, background noise will result. 
     Hybridization between sample DNA and appropriate ASO probes (i.e. labelled allele specific oligonucleotides of approximately the same length) can be carried out in a hybridization buffer containing an agent that eliminates disparities in the melting temperature of the ASOs used. One preferred agent is a quaternary ammonium salt (e.g. tetramethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium fluoride or tetraethyl ammonium fluoride). 
     Tetramethyl ammonium chiodde (TMAC) has been found to be an especially preferred quaternary ammonium salt for use in the subject invention. When added to hybridization buffer in a concentration in the range of about 2-5 M and optimally about 3 M, TMAC has been found to eliminate disparities in the melting temperatures of multiple ASOs, allowing the stringency of hybridization to be controlled as a function of probe length alone regardless of GC content (Wood, W. I. Proc. Natl. Acad. Sci. USA 82:1585-1588 (1985)). In addition to being present in the hybridization buffer, the quaternary ammonium salt may also be present in the wash solution, that is used to remove unbound nucleotides following hybridization. Further, it has been reported that TMAC in the PCR mixture can dramatically reduce and even eliminate non-specific priming events, thereby enhancing the specificity of the reaction (Hung, T. et. al. Nucl. Acid Res. 18:4953 (1990)). 
     In order to increase the signal to noise ratio, hybridization and washes should be carried out under stringent conditions. In other words, the temperature at which the hybridization reaction is conducted should be as high as possible for the length of ASO being used. The appropriate stringency for a particular ASO pool can be determined empirically. As an example, for the 17 base pair ASOs used to probe the cystic fibrosis transmembrane regulator gene, hybridizations were carried out at 52° C. 
     It has been found that signal to noise ratios of hybridized mutation specific ASOs can be further increased by including cold (i.e. non-labelled), normal (i.e. wild-type) oligonucleotides or portions thereof to the hybridization reaction preferably in a concentration in the range of about 1-100 times the concentration of labelled ASO. Presumably, unlabelled normal oligonucleotides or nucleotide portions outcompete the mutation specific labelled ASOs, where normal sequence is present thereby reducing the degree of non-specific hybridization occurring between the mutation specific ASOs and the normal wild-type sequence. 
     Subsequent to a wash step, hybridization can be detected using a means which is appropriate for the particular label used. For example, if ASOs are labelled radioactively, hybridization can be detected using autoradiography. 
     The procedure described herein can be used to test large numbers of samples simultaneously for multiple mutations within a gene. In addition, it can be used to simultaneously analyze several different individuals who have different disease indications (e.g. cystic fibrosis, sickle cell anemia, β-thallasemia, Tay-Sachs, Gaucher&#39;s disease and cancers resulting from certain mutations in genes, such as the P-53 gene) in a single hybridization assay and yet achieve disease specific results. 
     The present invention will now be illustrated by the following examples, which are not Intended and should not be construed as being limited in any way. 
     EXAMPLE 1 
     Efficient Multi-Mutation Testing in the Cystic Fibrosis Transmembrane Regulator (CFTR) Gene 
     Preparation of Sample DNA from CF Patient Blood 
     Whole blood samples collected in high glucose ACD Vacutainers™ (yellow top) were centrifuged and the buffy coat collected. The white cells were lysed with two washes of a 10:1 (v/v) mixture of 14 mM NH 4  Cl and 1 mM NaHCO 3 , their nuclei were resuspended in nuclei-lysis buffer (10 mM Tris, pH 8.0, 0.4 M NaCl, 2 mM EDTA, 0.5% SDS, 500 ug/ml proteinase K) and incubated overnight at 37° C. Samples were then extracted with a one-fourth volume of saturated NaCl and the DNA was precipitated in ethanol. The DNA was then washed with 70% ethanol, dried, and dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). 
     Preparation of Sample DNA from Buccal Cells 
     Buccal cells were collected on a sterile cytology brush (Scientific Products #S7766-1A) or female dacron swab (Medical Packaging Corp. #DTS-100), by twirling the brush or swab on the inner cheek for 30 seconds. DNA was prepared from the cheek cells, immediately or after a period of storage at room temperature or 4° C.: the brush or swab was immersed in 600 ul of 50 mM NaOH contained in a polypropylene microcentrifuge tube, and vortexed. The tube, still containing the brush or swab, was heated at 95° C. for 5 min., after which the brush/swab was carefully removed, leaving behind any residual liquid in the tube. The DNA solution was then neutralized with 60 ul of 1 M Tris, pH 8.0, and vortexed again (Mayall, E. and Williams, C. J. Med. Genet. 27:658 (1990)). After preparation, the buccal cell DNA was stored at 4° C., and 10 ul was used in a 50 ul PCR reaction. 
     Sample Amplification and Dot Blotting 
     Patient DNA samples were amplified in duplicate by PCR (Saiki, R. K. et. al., Science 239:487-491 (1988)) in a Perkin-Elmer Cetus 9600 Thermocycler. Five primer sets were used simultaneously to amplify regions of exons 4, 10, 11, 20, and 21 in a 50 ul reaction volume containing 200 ng of sample DNA and the following components; 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin, 200 uM in each dNTP, 0.4 uM in each amplification primer, and 2.5 units of Taq polymerase enzyme (Multiplex conditions were slightly modified by using 2.5 mM MgCl 2 , 5 units of Taq polymerase, and 10 ul of human genomic DNA prepared from cheek cells in a 50 ul reaction, or 1 ug of DNA prepared from blood). An initial denaturation of 20 sec. at 94° C. was done, followed by 28 cycles of amplification consisting of 10 sec. at 94° C., 10 sec. at 55° C., 10 sec. at 74° C., and a final soak at 74° C. for 5 min. Following amplification, 8 ul of the PCR product were electrophoresed on a 2% agarose gel to verify the presence of all five products and 8 ul of the mixed PCR products were added to 50 ul of denaturation solution (0.5 M NaOH, 2.0 M NaCl, 25 mM EDTA). The amplified products were spotted onto four nylon membranes (INC Biotrans), using a 96-well dot-blot apparatus (Bethesda Research Laboratories). The DNA was subsequently fixed to the membranes by baking the filters in vacuum at 80° C. for 15 min. 
     Hybridization and Washing 
       32  P-labeled mutation specific ASO probes: were made from the ASO sequences shown In Table 1. 
     
                                           TABLE 1                                 
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      ASO                    GC Content/ASO                               
Mutation                                                                  
      Sequence               %                                            
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G542X ATTCCACCTTCTCAAAG (SEQ ID NO:1)                                     
                             40                                           
G551D CTCGTTGATCTCCACTC (SEQ ID NO:2)                                     
                             53                                           
R553X CTCATTGACCTCCACTC (SEQ ID NO:3)                                     
                             53                                           
W1282X                                                                    
      CTTTCCTCCACTGTTGC (SEQ ID NO:4)                                     
                             47                                           
N1303K                                                                    
      TCATAGGGATCCAAGTT (SEQ ID NO:5)                                     
                             41                                           
Δ1507                                                               
      ACACCAAAGATATTTTC (SEQ ID NO:6)                                     
                             30                                           
1717-1                                                                    
      GGAGATGTCTTATTACC (SEQ ID NO:7)                                     
                             41                                           
R560T TATTCACGTTGCTAAAG (SEQ ID NO:8)                                     
                             53                                           
S549N CTCGTTGACCTCCATTC (SEQ ID NO:9)                                     
                             53                                           
R117H CGATAGAGTGTTCCTCC (SEQ ID NO:10)                                    
                             47                                           
621 + 1                                                                   
      GCAAGGAAGTATTACCT (SEQ ID NO:11)                                    
                             30                                           
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     Hybridizations were carried out in plastic bags containing pooled  32  p-labelled ASO probes shown in Table 1 and an excess of unlabeled normal sequences in a TMAC (Fisher Scientific) hybridization buffer (3.0 M TMAC, 0.6% SDS, 1.0 mM EDTA, 10 mM Na 3  PO 4 , pH 6.8, 5X Denhardt&#39;s solution, 40 ug/ml yeast RNA). ASO concentrations in the pools ranged from 0.03 to 0.15 pmol/ml of hybridization solution. The appropriate concentration for any particular ASO was determined empirically by testing the signal to noise ratio at an initial concentration of 0.03 and doubling the concentration until a proper signal to noise ratio was obtained. The bags were held overnight with agitation at 52° C. The membranes were then washed for 20 min. at room temperature with TMAC wash buffer (3.0 M TMAC, 0.6% SDS, 1.0ram EDTA, 10 mM Na 3  PO 4 , pH 6.8), followed by a second 20 min. TMAC wash at 52° C. The membranes were then dried and autoradiographs prepared by exposure to Kodak X-OMAT AR X-ray film. 
     Results 
     TMAC Properties and Hybridization Conditions 
     The specificity of hybridization in the presence of TMAC was established by probing amplified samples from individuals of known genotype with 11 of the 12 mutation-specific ASOs described above. Each ASO hybridized specifically only to samples carrying the complementary mutant sequence and not to samples which did not carry the complementary mutant sequence. Furthermore, the presence of TMAC in the hybridization and wash solutions allowed all of the hybridizations and washes to be performed at the same temperature (52° C.), despite a range in G-C content from 30% to 53% (Table 1). 
     CF/12 Test Design 
     Pools of ASOs were generated for more efficient mutation analysis. The compositions of the pools were determined by the total number of ASOs to be used in the assay, and by the estimated frequency of each mutation among Caucasian CF patients of northern European decent (Table 2). The frequency of each mutation determined how often it was necessary to follow up a pool-positive result with individual ASO hybridizations. 
     
                       TABLE 2                                                     
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Pool Design Considerations                                                
1)  Number of ASOs (N = 12)                                               
2)  Mutation Frequencies                                                  
Mutation       # of CF Chromosomes                                        
                               Frequency                                  
______________________________________                                    
Δ508     548             72.0%                                      
G551D          15              2.0%                                       
G542X          13              1.7%                                       
W1282X         15              2.0%                                       
N1303K         12              1.5%                                       
R553X          9               1.1%                                       
621 + 1        9               1.1%                                       
R117H          3               0.4%                                       
1717 - 1       3               0.4%                                       
R560T          2               0.3%                                       
Δ1507    1               0.1%                                       
S549N          1               0.1%                                       
Unknown        133             17.0%                                      
Total          764                                                        
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Conclusion (4 hybridizations)                                             
Filter #  1          2       3       4                                    
______________________________________                                    
          Wild-type  Δ508                                           
                             G551 D  621 + 1                              
          at 508             G542X   R117H                                
          position           W1282X  1717 - 1                             
                             N1303K  R560T                                
                             R553X   Δ507                           
                                     S549N                                
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 *Minimal Number of Independent Hybridizations                            
 
    
     Based on the foregoing considerations, analysis of the 12 mutations was broken into four individual hybridizations. As seen in Table 2, one of four identical filters was hybridized with a probe for the normal sequence at 508 position, with a second filter being hybridized only with a probe for the delta F508 mutation. A third was hybridized with a pool of ASOs for the five most frequent CFTR mutations after ΔF508:G551 D, G542X, W1282X, N1303K, and R553X. The fourth filter was probed with the remaining six mutations: 621+1, R117H, 1717-1, R560T, A1507, and S549N. The use of one filter for the normal 508 sequence and one for the ΔF508 mutation permitted immediate identification of all individuals affected with the most common mutation, as well as heterozygotes, these being the most widespread genotypes. Results from all four hybridizations were read in a binary manner. Each patient was represented by a pair of results, as samples were run in duplicate. Two critical quality-control Issues were addressed with this assay design. First, all samples were processed in duplicate, beginning with independent DNA extractions, followed by duplicate amplifications and analysis. This ensured against sample confusion at any of the sample-transfer steps. Second, each of the four independent filters contained a row of control samples of known genotype to ensure that each ASO within the respective pool had hybridized to its complimentary mutant sequence. These were the same samples used to determine the specificities of the ASOs. 
     Mutation Identification by Independent Hybridizations 
     Pool-positive samples were subsequently hybridized independently with the relevant ASOs to identify the specific mutation or mutations involved. Samples from patients with clinical indications of CF that were positive for only one of the pooled ASOs were then hybridized with the corresponding normal sequence to establish or exclude homozygosity for the mutation. 
     Validation Study 
     The use of pooled ASOs was validated by analyzing 382 DNA samples from CF-affected individuals, thereby obtaining data on 764 CFTR alleles. Secondary independent hybridizations of all pool-positive samples demonstrated that pool-positive results from these samples were due to the presence of one of the 12 mutations and not to non-specific hybridization. The detection frequencies observed in this study for the 12 mutations are given in Table 2. 
     Pool Complexity 
     For CFTR gene carder analysis in a clinical lab, there is a rationale for limiting the pools of ASOs to 5 or 6: it is necessary to be able subsequently to specify which ASO hybridized to a given positive sample. However, for other applications of the TMAC methodology, larger pools may be of great value. Therefore, the degree of pool complexity which could be attained pooling 11 of the 12 ASOs used in this study was tested. This pool was hybridized to a filter containing positive and negative control samples. The positive control samples were detected using the pool of probes. The negative control samples exhibited no significant non-specific hybridization. In other experiments, 14 ASOs have been hybridized simultaneously without non-specific hybridization. 
     EXAMPLE 2 
     Efficient Multi-Mutation Testing in the CFTR Gene and Gene for Sickle Cell Anemia 
     The same protocol was followed as in Example 1, except that into the Δ508 hybridization (Table 2) was added an allele specific oligonucleotide probe &#34;A&#34; (wild-type) made from an ASO having the sequence: CTCCTCAGGAGTCAGGT (SEQ ID NO:12) complementary to the normal, wild-type sequence within the β-globin gene and oligonucleotide probes &#34;S&#34; made from an ASO having the sequence: CTCCACAGGAGTCAGGT (SEQ ID NO:13) and &#34;C&#34; made from an ASO having the sequence: CTCCTTAGGAGTCAGGT (SEQ ID NO:14) were added to hybridizations #3 and #4 respectively, (Table 2). This allowed individual samples suggestive of containing a mutation within the β-globin gene (Chromosome 11) to be analyzed simultaneously in the same hybridization as individuals suggestive of carrying a mutation in the CFTR gene (Chromosome 7). 
     Equivalents 
     Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation; many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. 
     
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ATTCCACCTTCTCAAAG17                                                       
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CTCGTTGATCTCCACTC17                                                       
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CTCATTGACCTCCACTC17                                                       
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CTTTCCTCCACTGTTGC17                                                       
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TCATAGGGATCCAAGTT17                                                       
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ACACCAAAGATATTTTC17                                                       
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GGAGATGTCTTATTACC17                                                       
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TATTCACGTTGCTAAAG17                                                       
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CTCGTTGACCTCCATTC17                                                       
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CGATAGAGTGTTCCTCC17                                                       
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GCAAGGAAGTATTACCT17                                                       
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CTCCTCAGGAGTCAGGT17                                                       
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CTCCACAGGAGTCAGGT17                                                       
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CTCCTTAGGAGTCAGGT17                                                       
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