A method of PCR-based enrichment of a target substrate comprising the steps of: (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a plurality of different target-specific primer pairs for amplifying a plurality of different loci of the nucleic acid substrate, a universal primer, a DNA polymerase, and dNTPs, wherein each of the plurality of different target-specific primer pairs comprise a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise a 3' complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, and wherein the forward primer and the reverse primer each further comprise a 5' terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5' terminal sequence comprises a universal adaptor sequence, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence, and wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of each of the plurality of different target-specific primer pairs; (ii) exposing the PCR reaction mixture to at least two initial PCR cycles comprising a first annealing temperature for a first annealing duration to generate a plurality of different target specific amplicons, wherein each of the plurality of different target specific amplicons comprise the 5' terminal sequence at their 5' terminal region; (iii) exposing the PCR reaction mixture to an appropriate number of additional PCR cycles to further amplify the plurality of different target specific amplicons. (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a plurality of different target-specific primer pairs for amplifying a plurality of different loci of the nucleic acid substrate, a universal primer, a DNA polymerase, and dNTPs, wherein each of the plurality of different target-specific primer pairs comprise a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise a 3' complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, and wherein the forward primer and the reverse primer each further comprise a 5' terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5' terminal sequence comprises a universal adaptor sequence, wherein the universal primer comprises a modified universal adaptor sequence, wherein the modified universal adaptor sequence possesses a modified base, wherein the modified base targets cleavage of the universal primer by an endonuclease, wherein the modified universal adaptor sequence and the universal adaptor sequence are complementary to a common sequence, and wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of each of the plurality of different target-specific primer pairs; (ii) exposing the PCR reaction mixture to at least two initial PCR cycles comprising a first annealing temperature for a first annealing duration to generate a plurality of different target specific amplicons, wherein each of the plurality of different target specific amplicons comprise the 5' terminal sequence at their 5' terminal region; (iii) exposing the PCR reaction mixture to an appropriate number of additional PCR cycles to further amplify the plurality of different target specific amplicons.