Real-time polymerase chain reaction using large target amplicons

A method for analyzing a target nucleic acid sequence in a biological material, said method comprising:

• (i) adding to said biological material an effective amount of at least two nucleic acid primers,
• wherein said nucleic acid primers hybridize under stringent conditions to predetermined nucleic acid sequences of said target nucleic acid sequence that are separated by at least about 750 nucleic acid residues,
• (ii) amplifying said target nucleic acid sequence by polymerase chain reaction, said polymerase chain reaction comprising adding a polymerase and to said biological material and primers to form an amplification mixture and thermally cycling said amplification mixture between at least one denaturation temperature and at least one elongation temperature,
• wherein said elongation temperature is not more than about 70□ C and said denaturation temperature is not more than about 95□ C, and further wherein during each thermal cycle said amplification mixture is maintained at said denaturation temperature for a period of not more than about 30 seconds and at said elongation temperature for a period of not less than about 1 minute;
• and
• (iii) detecting and quantifying said target nucleic acid sequence.