Method for polymerase chain reactions with use of a DNA polymerase with proofreading properties

A method for nucleic acid amplification, comprising amplifying a nucleic acid via performing a polymerase chain reaction (PCR) in the presence of (i) a DNA template, (ii) a DNA polymerase with proofreading activity, (iii) at least one primer, (iv) deoxyribonucleoside triphosphates, and (v) at least one target substrate,

• wherein the at least one primer is not resistant to degradation by the proofreading activity of the DNA polymerase,
• wherein the at least one target substrate
• (a) is a single stranded oligonucleotide in which at least one phosphate in the backbone of said single stranded oligonucleotide is replaced by at least one phosphorothioate,
• (b) is modified at its 3′-end so that it is incapable of being elongated by the DNA polymerase with proofreading activity,
• (c) has a random sequence,
• (d) forms a double stranded nucleic acid molecule with a strand of the DNA template under PCR conditions, and
• (e) is able as the double stranded nucleic acid molecule of (d) to serve as a binding site for the polymerase with proofreading activity, thereby reducing the 3′-exonuclease activity of the DNA polymerase toward the primer or the DNA template, and
• wherein the molecular ratio of the polymerase with proofreading activity to the at least one target substrate is between 1:1 and 1:1000.