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 detect
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.
대표청구항▼
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 com
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, andiv) 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; andiii) 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 25th 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 25th percentile for age-matched controls; and(d) administering to the subject diagnosed at higher cardiovascular risk: i) a modified statin therapy; and/orii) a second therapeutic agent known to treat cardiovascular disease.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.