Methods for full-length amplification of double-stranded linear nucleic acids of unknown sequences

摘要

An adaptor for use in amplifying all linear, double-stranded nucleic acid molecules of unknown sequences in a sample is disclosed. The adaptor consists of: (1) the first oligonucleotide (P- oligo) with a phosphate at the 5'end and without an additional thymine nucleotide at the 3' end; and (2) the second oligonucleotide (T~oligo) with an extra 3'-T and without a 5'-phosphate. The P-oligo and T-oligo are complementary to each other except at the 3' -T (thymine) in the T-oligo. The adaptor is ligated to nucleic acids of unknown sequences which have an extra A in the 3' end (3 ' ~A overhang) to form adaptor-ligated target nucleic acids. The T-oligo is then employed as a single primer for T-oligo-primed polymerase chain reaction (TOP-PCR) and amplifies the nucleic acids of unknown sequences in full-length.