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; 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;
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; and
d) determining the average length or abundance of telomeric DNA in the sample, wherein the average length or abundance of telomeric DNA in the sample is determined by: i) determining a concentration of the first, second and third amplicon by comparison to a control polymerase reaction; (ii) determining a ratio of the concentration of the first amplicon to an average concentration of the second and third amplicons; and (iii) converting the ratio from step (ii) to base pairs of telomere sequence per genome.
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.
4. The method of claim 1 , wherein the first reverse primer is blocked from priming the first target nucleic acid.
5. The method of claim 4 , wherein the first reverse primer is blocked from priming the first target nucleic acid by a terminal 3′ mismatched base.
6. The method of claim 1 , wherein the second target nucleic acid is within a gene of known copy number.
7. The method of claim 6 , wherein the gene of known copy number is a low copy number gene.
8. The method of claim 6 , wherein the second target nucleic acid is a single copy number gene.
9. 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.
10. 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.
11. The method of claim 10 , wherein the second detection label further comprises an oligonucleotide comprising the sequence of SEQ ID NO: 5.
12. 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.
13. 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.
14. The method of claim 13 , wherein the step of obtaining a chromosomal DNA sample comprises isolating one or more cell type from a fluid 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.
15. The method of claim 1 , wherein the concentration of first, second, and third amplicon are determined by comparison to a control reference DNA.
16. 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.