Disease detection by digital protein truncation assays

Abstract

Genetic diseases can be diagnosed by detection of mutations in causative genes. Protein truncation assays can be used to detect gene products of truncation-type mutations. However, the sensitivity of the assays is often insufficient to detect mutations present in a sample of DNA at a low frequency. Sensitivity can be increased by dividing samples so that the signal generated by a mutant allele comprises a larger fraction of the total alleles than prior to dividing. Thus a previously undetectable signal generated by the mutant allele can become detectable in the assay. Such increased sensitivity permits detection at early stages and in samples having high levels of other alleles.

1 . A method of detecting tumors, comprising: 
 dividing a test sample of APC alleles isolated from a patient, to form a plurality of aliquots of APC alleles;    amplifying said APC alleles in said plurality of aliquots to form amplified APC alleles;    transcribing and translating proteins in vitro using said amplified APC alleles as transcription templates;    determining size or composition of said proteins, wherein proteins which differ in size or composition from the protein produced by a wild-type APC allele indicate a mutation in an amplified APC allele which indicates a tumor in the patient.    
     
     
         2 . The method of  claim 1  further comprising the step of determining the concentration of APC alleles in the test sample by limiting dilution polymerase chain reaction.  
     
     
         3 . The method of  claim 1  wherein said proteins are subjected to gel electrophoresis.  
     
     
         4 . The method of  claim 1  wherein composition of said proteins is determined using mass spectroscopy.  
     
     
         5 . The method of  claim 1  wherein said aliquots comprise on average between 0 and 20 APC alleles prior to amplification.  
     
     
         6 . The method of  claim 1  wherein said aliquots comprise on average between 0 and 10 APC alleles prior to amplification.  
     
     
         7 . The method of  claim 1  wherein said aliquots comprise on average between 0 and 5 APC alleles prior to amplification.  
     
     
         8 . The method of  claim 1  wherein said aliquots comprise on average between 0 and 1 APC alleles prior to amplification.  
     
     
         9 . The method of  claim 1  wherein said aliquots comprise on average between 1 and 20 APC alleles prior to amplification.  
     
     
         10 . The method of  claim 1  wherein said aliquots comprise on average between 5 and 20 APC alleles prior to amplification.  
     
     
         11 . The method of  claim 1  wherein said aliquots comprise on average between 10 and 20 APC alleles prior to amplification.  
     
     
         12 . The method of  claim 1  wherein said aliquots comprise on average between 1 and 5 APC alleles prior to amplification.  
     
     
         13 . The method of  claim 1  wherein said aliquots comprise on average between 2 and 4 APC alleles prior to amplification.  
     
     
         14 . The method of  claim 1  wherein size is determined by polyacrylamide gel electrophoresis.  
     
     
         15 . The method of  claim 1  wherein the step of dividing is performed by diluting the APC alleles to achieve an average number of APC alleles per aliquot of between 0 and 20.  
     
     
         16 . The method of  claim 1  wherein the step of dividing is performed by diluting the captured APC alleles to achieve an average number of APC alleles per aliquot of between 0 and 5.  
     
     
         17 . The method of  claim 1  wherein the test sample is a body fluid or exudate.  
     
     
         18 . The method of  claim 1  wherein the test sample is a washing or aspirate of an organ.  
     
     
         19 . The method of  claim 1  wherein the test sample of APC alleles are isolated from a stool sample of the patient.  
     
     
         20 . The method of  claim 1  wherein the step of dividing is performed by diluting the test sample.  
     
     
         21 . The method of  claim 19  wherein at least 500 bp of template is amplified.  
     
     
         22 . The method of  claim 19  wherein at least 750 bp of template is amplified.  
     
     
         23 . The method of  claim 19  wherein at least 1 kb of template is amplified.  
     
     
         24 . The method of  claim 1  wherein at least a portion of exon 15 is amplified.  
     
     
         25 . The method of  claim 19  wherein codons 1210 through 1581 of APC are amplified.  
     
     
         26 . The method of  claim 1  wherein the step of amplifying employs a first and a second set of primers, wherein the first set of primers amplifies a template to which the second set of primers is complementary.  
     
     
         27 . The method of  claim 1  wherein antibodies to a C-terminal epitope of said proteins are used to determine composition of said proteins.  
     
     
         28 . The method of  claim 1  wherein antibodies to a C-terminal epitope of said proteins are used to immnunodeplete said proteins of full-length proteins.  
     
     
         29 . The method of  claim 1  wherein antibodies to an N-terminal epitope of said proteins are used to determine size or composition of said proteins.  
     
     
         30 . A method of detecting a disease associated with a mutation in a gene, comprising: 
 dividing a test sample of alleles of the gene isolated from a patient, to form a plurality of aliquots of alleles of the gene;    amplifying said alleles in said plurality of aliquots to form amplified alleles;    transcribing and translating proteins in vitro using said amplified alleles as transcription templates;    determining size or composition of said proteins, wherein proteins which differ in size or composition from the protein produced by a wild-type allele of the gene indicate a mutation in an amplified allele of the gene which indicates the disease in the patient.    
     
     
         31 . The method of  claim 30  wherein the step of dividing is performed by diluting the test sample.  
     
     
         32 . The method of  claim 30  further comprising the step of determining the concentration of said alleles in the test sample by limiting dilution polymerase chain reaction.  
     
     
         33 . The method of  claim 30  wherein said aliquots comprise on average between 0 and 20 alleles prior to amplification.  
     
     
         34 . The method of  claim 30  wherein said aliquots comprise on average between 0 and 10 alleles prior to amplification.  
     
     
         35 . The method of  claim 30  wherein said aliquots comprise on average between 0 and 5 alleles prior to amplification.  
     
     
         36 . The method of  claim 30  wherein said aliquots comprise on average between 0 and 1 alleles prior to amplification.  
     
     
         37 . The method of  claim 30  wherein said aliquots comprise on average between 1 and 20 alleles prior to amplification.  
     
     
         38 . The method of  claim 30  wherein said aliquots comprise on average between 5 and 20 alleles prior to amplification.  
     
     
         39 . The method of  claim 30  wherein said aliquots comprise on average between 10 and 20 alleles prior to amplification.  
     
     
         40 . The method of  claim 30  wherein said aliquots comprise on average between 1 and 5 alleles prior to amplification.  
     
     
         41 . The method of  claim 30  wherein said aliquots comprise on average between 2 and 4 alleles prior to amplification.  
     
     
         42 . The method of  claim 30  wherein the step of dividing is accomplished by diluting the alleles to achieve an average number of alleles per aliquot of between 0 and 20.  
     
     
         43 . The method of  claim 30  wherein the step of dividing is accomplished by diluting the alleles to achieve an average number of alleles per aliquot of between 0 and 5.  
     
     
         44 . The method of  claim 30  wherein the gene is Merlin, and the disease is neurofibromatosis type 2.  
     
     
         45 . The method of  claim 30  wherein the gene is VHL, and the disease is von Hippel-Landau disease.  
     
     
         46 . The method of  claim 30  wherein the gene is CF, and the gene is cystic fibrosis.  
     
     
         47 . The method of  claim 30  wherein the gene is hMSH2, and the disease is Hereditary non-Polyposis Colon Cancer (HNPCC).  
     
     
         48 . The method of  claim 30  wherein the gene is hMLH1, and the disease is Hereditary non-Polyposis Colon Cancer (HNPCC).  
     
     
         49 . The method of  claim 30  wherein the gene is hPMS2, and the disease is Hereditary non-Polyposis Colon Cancer (HNPCC).  
     
     
         50 . The method of  claim 30  wherein said proteins are subjected to gel electrophoresis.  
     
     
         51 . The method of  claim 30  wherein composition of said proteins is determined using mass spectroscopy.  
     
     
         52 . The method of  claim 30  wherein size is determined by polyacrylamide gel electrophoresis.  
     
     
         53 . The method of  claim 30  wherein the test sample is a body fluid or exudate.  
     
     
         54 . The method of  claim 30  wherein the test sample is a washing or aspirate of an organ.  
     
     
         55 . The method of  claim 30  wherein at least 500 bp of template is amplified.  
     
     
         56 . The method of  claim 30  wherein at least 750 bp of template is amplified.  
     
     
         57 . The method of  claim 30  wherein at least 1 kb of template is amplified.  
     
     
         58 . The method of  claim 30  wherein the step of amplifying employs a first and a second set of primers, wherein the first set of primers amplifies a template to which the second set of primers is complementary.  
     
     
         59 . The method of  claim 30  wherein antibodies to a C-terminal epitope of said proteins are used to determine composition of said proteins.  
     
     
         60 . The method of  claim 30  wherein antibodies to a C-terminal epitope of said proteins are used to immunodeplete said proteins of full-length proteins.  
     
     
         61 . The method of  claim 30  wherein antibodies to an N-terminal epitope of said proteins are used to determine size or composition of said proteins.