Digital polymerase chain reaction method for detecting nucleic acids in samples

A method for analyzing nucleic acids in a sample, said sample containing one or more linear, double stranded nucleic acid fragment(s) (NA fragment(s)), said method comprising the steps of:

• (a) subjecting the sample to a 3′-A tailing reaction allowing for adding an adenine nucleotide (A) to the 3′-tail of the NA fragment(s) to produce 3′-adenine nucleotide (3′-A) overhang nucleic acid fragment(s) (3′-A overhang NA fragment(s));
• (b) providing a 3′-thymine (T) or 3′-uracil (U) nucleotide overhang double-stranded homogenous adapter which comprises a P oligo strand carrying a 5′-phosphate for ligation to the 3′-A overhang NA fragment(s) and a T/U oligo strand carrying a 3′-T or 3′-U without a 5′-phosphate, wherein the T/U oligo strand is complimentary to the P oligo strand except at the 3′-T or 3′-U of the T/U oligo strand;
• (c) subjecting the sample of (a) to a ligation reaction allowing for ligating the homogenous adapter to the 3′-A overhang NA fragment(s) at both ends to produce adapter-ligated nucleic acid fragment(s) (adapter-ligated NA fragment(s));
• (d) combining the sample of (c) with polymerase chain reaction (PCR) reagents and detection reagents to provide amplification/detection-ready sample, wherein the PCR reagents include a single-type primer having the nucleic acid sequence of the T/U oligo strand for amplification and the detection reagents include one or more fluorescent probe(s) producing fluorescent signals and specifically hybridizing with the NA fragment(s) for detection;
• (e) partitioning the amplification/detection-ready sample of (d) into a plurality of partitions, each containing limited copy of the adapter-ligated NA fragment(s);
• (f) performing PCR in each partition using the adapter-ligated NA fragment(s) as template(s) and the single-type primer as both forward and reverse primers to amplify the adapter-ligated NA fragment(s);
• and
• (g) assessing the fluorescent signal(s) in each fraction.