AbstractA highly efficient chemical ligation was developed for quantitative conjugation of PNA with DNA (PNA or peptide) by using the copper‐catalyzed azide–alkyne cycloaddition reaction. Whereas PNAs with an alkyne at the C‐terminus and an azide at the N‐terminus have been used, an efficient click–click reaction occurs. The PNA click ligation is sequence specific and capable of single nucleotide discrimination.