By S Nadkarni
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Polymerization complete INTRODUCTION 19 (1992, 1993) pioneered the use of ethidium bromide to quantify PCR products as they accumulate. Amplification produces increasing amounts of double-stranded DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence versus cycle number it is possible to analyse the PCR kinetics in real time. This is much more satisfactory than analyzing product accumulation after a fixed number of cycles. The principal draw back to the use of ethidium bromide is that both specific and non-specific products generate a signal.
Occasionally, a polymerase adds to the growing chain an incorrect nucleotide, which cannot form a proper base pair with the base in the template strand. lired nucleotide activates the 3' -5' exonuclease activity, which cleaves the unpaired nucleotide from the 3' -OH end of the growing chain. Because it cleaves off an incorrect nucleotide and gives the polymerase another chance to get it right, the 3' ~ 5' exonuclease activity of DNA polymerase is also called the editing function or proofreading function.
Polymerase I plays an essential but secondary, role in replication that will be described in a later section. Eukaryotic cells also contain several DNA polymerases. The enzyme responsible for the replication of chromosomal DNA is called polymerase a, Mitochondria have their own DNA polymerase to replicate the mitochondrial DNA. In addition to their ability to polymerize nucleotides, most DNA polymerase are capable of nucleases activities that break phosphodiester bonds in the sugar-phosphate backbones of nucleic acid chains.