Multi-copy reference assay

Abstract

A method, comprising amplifying a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject; amplifying a reference nucleic acid sequence in the sample; quantifying the amplified sequence of interest relative to the amplified reference sequence; and determining a copy number of the sequence of interest from the relative quantified amplified sequence of interest. The reference sequence may have at least 80% sequence identity to at least one of SEQ ID NO:1-38, such as SEQ ID NO:1-13. Also disclosed are kits and compositions, each comprising a first probe which specifically hybridizes to at least a portion of at least one reference sequence. Also disclosed is a system configured to perform the above method.

What is claimed is: 
     
         1 . A method, comprising:
 amplifying a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject;   amplifying a reference sequence in the sample, wherein the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs,
 wherein the at least one portion is present in chr1-121790-133586, chr1-329448-341534, chr1-648129-660266, chr1-222643865-228172047, chr1-243203764-243215874, chr10-38741930-38753964, chr11-114010-126106, chr16-90239446-90251554, chr19-183944-196032, chr2-114323560-114323652, chr2-243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4-119557144-120325498, chr4-165196360-165199636, chr5-180756063-180768074, chr6-170921836-170922549, chr7-39837560-63231088, chr7-128296352-128298474, chr8-143133-150475, chr9-49679-49771, chrY-26424506-27537936, or chr6-132951-145064; 
 the at least one portion is present in at least a first minimum number of copies in the genome; and 
 at least one copy of the at least one portion is present on each of at least a second minimum number of chromosomes; 
   quantifying the amplified sequence of interest relative to the amplified reference sequence; and   determining a copy number of the sequence of interest from the relative quantified amplified sequence of interest.   
     
     
         2 . The method of  claim 1 , wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NOs: 1-31. 
     
     
         3 . The method of  claim 1 , wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO:1-13. 
     
     
         4 . The method of  claim 1 , wherein the sample comprises tissue suspected of being cancer tissue. 
     
     
         5 . The method of  claim 4 , wherein the sample has been subjected to formalin fixing and paraffin embedding (FFPE) prior to amplifying the sequence of interest and amplifying the reference sequence. 
     
     
         6 . The method of  claim 4 , further comprising:
 diagnosing the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.   
     
     
         7 . The method of  claim 1 , wherein amplifying the sequence of interest and amplifying the reference sequence are performed by TaqMan quantitative polymerase chain reaction (qPCR). 
     
     
         8 . A composition, comprising:
 a first probe which specifically hybridizes to at least a portion of at least one reference sequence having at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chr1-121790-133586, chr1-329448-341534, chr1-648129-660266, chr1-222643865-228172047, chr1-243203764-243215874, chr10-38741930-38753964, chr11-114010-126106, chr16-90239446-90251554, chr19-183944-196032, chr2-114323560-114323652, chr2-243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4-119557144-120325498, chr4-165196360-165199636, chr5-180756063-180768074, chr6-170921836-170922549, chr7-39837560-63231088, chr7-128296352-128298474, chr8-143133-150475, chr9-49679-49771, chrY-26424506-27537936, or chr6-132951-145064;   the at least one portion is present in at least a first minimum number of copies in the genome; and   at least one copy of the at least one portion is present on each of at least a second minimum number of chromosomes.   
     
     
         9 . The composition of  claim 8 , wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO:1-31. 
     
     
         10 . The composition of  claim 8 , wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO:1-13. 
     
     
         11 . The composition of  claim 8 , wherein the first probe comprises a nucleic acid sequence configured to specifically hybridize to at least the portion of the at least one reference sequence, a fluorescent reporter at a first end of the nucleic acid sequence, and a fluorescent quencher at a second end of the nucleic acid sequence. 
     
     
         12 . The composition of  claim 8 , further comprising:
 a first primer configured to specifically hybridize to a first end of the at least one reference sequence, and   a second primer configured to specifically hybridize to a sequence complementary to a second end of the at least one reference sequence.   
     
     
         13 . The composition of  claim 8 , further comprising:
 a second probe which specifically hybridizes to at least a portion of at least one nucleic acid sequence of interest.   
     
     
         14 . The composition of  claim 13 , further comprising:
 a third primer configured to specifically hybridize to a first end of the at least one nucleic acid sequence of interest, and   a fourth primer configured to specifically hybridize to a sequence complementary to a second end of the at least one nucleic acid sequence of interest.   
     
     
         15 . A system, comprising:
 a nucleic acid amplifier configured to amplify a nucleic acid sequence of interest in a sample comprising genomic DNA of a subject and amplify a reference sequence in the sample,   a reagent reservoir containing at least a first primer configured to specifically hybridize to a first end of the at least one reference sequence, wherein the reference sequence has at least 80% sequence identity to at least one portion of genomic DNA comprising from about 60 to about 150 base pairs, wherein the at least one portion is present in chr1-121790-133586, chr1-329448-341534, chr1-648129-660266, chr1-222643865-228172047, chr1-243203764-243215874, chr10-38741930-38753964, chr11-114010-126106, chr16-90239446-90251554, chr19-183944-196032, chr2-114323560-114323652, chr2-243064480-243071940, chr20-62921559-62933673, chr3-197950387-197962431, chr4-119557144-120325498, chr4-165196360-165199636, chr5-180756063-180768074, chr6-170921836-170922549, chr7-39837560-63231088, chr7-128296352-128298474, chr8-143133-150475, chr9-49679-49771, chrY-26424506-27537936, or chr6-132951-145064; the at least one portion is present in at least a first minimum number of copies in the genome; and at least one copy of the at least one portion is present on each of at least a second minimum number of chromosomes; , and a second primer configured to specifically hybridize to a sequence complementary to a second end of the at least one reference sequence;   a detector configured to provide a first indication relating to an amount of the amplified sequence of interest and a second indication relating to an amount of the amplified reference sequence; and   a controller configured to quantify the amplified sequence of interest relative to the amplified reference sequence, based at least in part on the first indication and the second indication; and determine a copy number of the sequence of interest from the relative quantified amplified sequence of interest.   
     
     
         16 . The system of  claim 15 , wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO:1-31. 
     
     
         17 . The system of  claim 16 , wherein the reference sequence has at least 80% sequence identity to at least one of SEQ ID NO:1-13. 
     
     
         18 . The system of  claim 17 , wherein the sample comprises tissue suspected of being cancer tissue. 
     
     
         19 . The system of  claim 18 , wherein the sample has been subjected to formalin fixing and paraffin embedding (FFPE). 
     
     
         20 . The system of  claim 19 , wherein the controller is further configured to indicate the subject as having a cancer-related biomarker, based on the sequence of interest being associated with the cancer and the copy number being indicative of the cancer.