Method for amplifying free nucleic acids directly from one drop of unpurified sample

A method for amplifying free nucleic acids directly from an unpurified sample, wherein the unpurified sample comprises nucleic acid fragments, the method comprising the sequential steps of:

• (a) dilution:
• mixing the unpurified sample with a buffer thoroughly to form a mixed solution;
• (b) protein denaturation:
• adding the mixed solution into a test tube, heating the mixed solution in the test tube to denature proteins in the mixed solution, and then cooling the mixed solution to room temperature;
• (c) end modification of nucleic acid molecules:
• subjecting the nucleic acid fragments in the mixed solution to a processing reaction required for an adapter-dependent polymerase chain reaction (PCR);
• (d) adapter ligation:
• performing a ligation reaction between the processed nucleic acid fragments and double-stranded adapters having a single type of sequence, defined as double-stranded homogeneous adapters, in order to form nucleic acid fragments each ligated at each of two ends thereof with one said double-stranded homogeneous adapter, wherein each said double-stranded homogeneous adapter is a complementary double-stranded nucleic acid (dsNA) fragment with one strand being an oligonucleotide carrying a 5′-phosphate group and the other strand being an oligonucleotide carrying a thymine (T) or uracil (U);
• and
• (e) PCR:
• adding components required for performing the adapter-dependent PCR on the ligated nucleic acid fragments, and then performing the adapter-dependent PCR on the ligated nucleic acid fragments in the mixed solution.