Multiplex quantitative pcr

Abstract

Disclosed are methods and compositions for determining the average length or abundance of a first target nucleic by calculating the abundance of a first target nucleic acid (T) relative to the average abundance (S) of a second and a third target nucleic acid, in a single well using a separate detection label for each target nucleic acid. In various aspects, the first target nucleic acid is a telomere. In exemplary aspects, the disclosed methods and compositions can be used to determine the average telomere length in a biological sample. The average telomere length determined using the disclosed methods and compositions can be correlated to a variety of clinically important conditions and indices. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

What is claimed is: 
     
         1 . A method for determining average telomere length or abundance, comprising:
 (a) contacting a first target nucleic acid with a first primer set, a second target nucleic acid with a second primer set, and a third target nucleic acid target with a third primer set;
 i) wherein the first primer set comprises a first forward primer and a first reverse primer; 
 ii) wherein the second primer set comprises a second forward primer and a second reverse primer; 
 iii) wherein the third primer set comprises a third forward primer and a third reverse primer, and 
 iv) wherein the first target nucleic acid comprises a telomere repeat sequence; 
   (b) amplifying by polymerase chain reaction the first target nucleic acid with the first primer set to form a first amplicon, the second target nucleic acid with the second primer set to form a second amplicon, and the third target nucleic acid with the third primer set to form a third amplicon;   (c) determining during the polymerase chain reaction the amount of the first, second, and third amplicons:
 i) wherein the first amplicon is detected using a first detection label; 
 ii) wherein the second amplicon is detected using a second detection label; and 
 iii) wherein the third amplicon is detected using a third detection label; 
   (d) determining the average length or abundance of telomeric DNA in the sample.   
     
     
         2 . The method of  claim 1 , wherein each of the first forward primer and a first reverse primer comprise:
 (a) a 3′ portion that hybridizes to a telomeric repeat sequence under annealing conditions; and   (b) a 5′ portion having an anchor sequence that does not hybridize to a telomeric repeat sequence.   
     
     
         3 . The method of  claim 1 , wherein the first reverse primer is a mismatch primer comprising at least one mismatched nucleotide adjacent to or including the 3′ end of the primer; and wherein the at least one mismatched nucleotide is not complementary to the target nucleic acid, but is complementary to the 3′ terminal nucleotide of the first forward primer. 
     
     
         4 . The method of  claim 3 , wherein the first forward primer comprises the sequence of SEQ ID No.: 1; and wherein the first reverse primer comprises the sequence of SEQ ID No.: 2. 
     
     
         5 . The method of  claim 1 , wherein the first reverse primer is blocked from priming the first target nucleic acid. 
     
     
         6 . The method of  claim 5 , wherein the first reverse primer is blocked from priming the first target nucleic acid by a terminal 3′ mismatched base. 
     
     
         7 . The method of  claim 1 , wherein the second target nucleic acid is within a gene of known copy number. 
     
     
         8 . The method of  claim 7 , wherein the gene of known copy number is a low copy number gene. 
     
     
         9 . The method of  claim 7 , wherein the second target nucleic acid is a single copy number gene. 
     
     
         10 . The method of  claim 1 , wherein the second forward primer comprises SEQ ID NO.: 3; and wherein the second reverse primer comprises SEQ ID NO.: 4. 
     
     
         11 . The method of  claim 1 , wherein each of the first detection label, second detection label, and third detection label independently comprise fluorogenic moieties; and wherein each of the fluorogenic moieties is detectable separably and simultaneously. 
     
     
         12 . The method of  claim 11 , wherein the second detection label further comprises an oligonucleotide comprising the sequence of SEQ ID NO.; 5. 
     
     
         13 . The method of  claim 1 , wherein the second amplicon is from about 50 to about 250 bp in length; and wherein the third amplicon is from about 50 to about 250 bp in length. 
     
     
         14 . The method of  claim 1 , further comprising the step of obtaining a chromosomal DNA sample prior to contacting the first, second, and third target nucleic acids with the first, second, and third primer sets, respectively; and wherein the chromosomal DNA sample comprises the first, second, and third target nucleic acids. 
     
     
         15 . The method of  claim 14 , wherein the step of obtaining a chromosomal DNA sample comprises isolating one or more cell type from a liquid sample obtained a subject; and wherein the cell type isolated comprise circulating tumor cells, circulating stem cells, lymphocytes, granulocytes, myeloid cells, neutrophils, monocytes, macrophages, platelets, and leukocytes. 
     
     
         16 . The method of  claim 1 , wherein the concentration of first, second, and third amplicon are determined by comparison to a control reference DNA. 
     
     
         17 . The method of  claim 1 , wherein determining the average length or abundance of the first amplicon comprises the steps:
 (a) determining the concentration of the first, second, and third amplicon by comparison to a control polymerase chain reaction;   (b) determine the ratio of the concentration of the first amplicon to the average or weighted concentration of the second and third amplicons; and   (c) converting the ratio from step (b) to base pairs of telomere sequence per genome.   
     
     
         18 . A method for allogeneic transplant hematopoietic stem cell donor selection, the method comprising:
 (a) obtaining samples from one or more HLA-matched potential donor subjects;   (b) determining the average length or abundance of telomeric DNA for each of the HLA-matched donor subjects by the method of  claim 1 ;   (c) identifying one or more donor subjects with a first amplicon average length or abundance that in the upper 25 th  percentile for age-matched controls;   (d) obtaining a transplantable hematopoietic stem cell sample from the identified donor subject; and   (e) transplanting the hematopoietic stem cell sample to a recipient subject.   
     
     
         19 . The method of  claim 18 , wherein the recipient subject has been diagnosed with a cancer, cardiovascular disease, or with a need for a bone marrow transplant. 
     
     
         20 . A method for reclassification of cardiovascular disease risk, the method comprising:
 (a) obtaining a sample a subject, wherein the subject has been diagnosed to meet 2013 ACC/AHA Guideline on the Treatment of Blood Cholesterol criteria for low-intensity statin therapy;   (b) determining the average length or abundance of the first amplicon in the sample for by the method of claim;   (c) diagnosing the subject at higher cardiovascular risk when the sample has been determined to have with a first amplicon average length or abundance that in the lower 25 th  percentile for age-matched controls; and   (d) administering to the subject diagnosed at higher cardiovascular risk:
 i) a modified statin therapy; and/or 
 ii) a second therapeutic agent known to treat cardiovascular disease.