Abstract: Disclosed is a method wherein an unknown DNA sequence 3′ to a known DNA sequence is amplified specifically in a first round of PCR using a 5′-blocked primer which hybridizes to the known portion of the DNA sequence. The resultant population of complementary strands to the unknown region is melted to yield single-stranded DNA molecules which are complementary to the unknown DNA sequence, but having a known 5′ terminal sequence which is blocked to further reaction. The single-stranded DNA molecules are coupled at their 3′ termini to an arbitrary, 3′-blocked synthetic primer of known sequence to yield single-stranded DNA target molecules having a known sequence at both the 5′ and 3′ termini and an unknown sequence therebetween. Amplification of the DNA target molecules, using primers complementary to the known 5′ and 3′ termini, causes specific amplification of the unknown DNA between the 5′ and 3′ termini of the DNA target molecules.