System and method for ligand-limited normalizing polymerase chain reaction (lln-pcr)

A method for obtaining a normalized quantity of DNA amplicons, the method comprising:

• adding, to a volume of a sample comprising a target DNA segment, a first concentration of a forward primer, wherein the forward primer is complementary to a first end of the target DNA segment;
• adding, to the volume of the sample, a second concentration of a second primer, wherein the second primer comprises an unlabeled reverse primer having a first annealing temperature, and a binding site specific to a complement of a third primer;
• adding, to the volume of the sample, a third concentration of the third primer, wherein the third primer is a ligand modified reverse primer complementary to a second end of the target DNA segment and comprises a normalizing ligand,
• wherein the third primer has a second annealing temperature, which is greater than the first annealing temperature,
• wherein the third concentration is less than the second concentration;
• and
• performing a polymerase chain reaction (PCR) on the volume of the sample to obtain a normalized concentration of ligand-tagged amplicons, wherein the PCR comprises one or more melt cycles, annealing cycles, and extension cycles,
• wherein the annealing cycles are performed at a third annealing temperature, which is closer to the second annealing temperature than to the first annealing temperature.