MULTIPLEX QUANTITATIVE PCR
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.
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.