High background and low specific product yield are serious problems in DNA amplification by PCR (Polymerase Chain Reaction) and PCR-related techniques. Based on preliminary data, the applicants propose a technique termed """"""""EnzyStart"""""""" to solve the problems. """"""""EnzyStart"""""""" incorporates oligonucleotide primers that are modified with a """"""""block"""""""" covalently bound to the oxygen at their 3-prime end and a new thermostable enzyme in the routine PCR reaction mixture. The """"""""block"""""""" at the 3-prime end of the primers inhibits their extension by DNA polymerase at any temperature if the 3-prime block is not removed. The thermostable enzyme specifically and efficiently removes the 3-prime block only at temperatures higher than 55 degrees C. The applicants state that the combination of these two elements eliminates nonspecific primer extension during the reaction setup at room temperature and prevents the amplification of non-target DNA sequence at temperatures lower than 55 degrees C during the first cycle. The applicants also state that success in these endeavors will provide a general, reliable, and inexpensive technique to increase the specificity and sensitivity of PCR and PCR-related techniques for biomedical research such as the Human Genome Project, cancer research, pathogen detection and clinical diagnosis.
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