|MadSci Network: Molecular Biology|
The length of a section of DNA that can be amplified by a PCR reaction is limited by the processivity of the DNA polymerase used. Processivity is a measure of the average number of nucleotide residues a polymerase molecule adds to a growing polynucleotide before it dissociates from the complex. For Taq polymerase, a commonly used polymerase for PCR, the enzyme’s processivity limits one to replicating sequences of under 10 kb. The length of DNA that can be replicated will also depend upon also the complexity of the target DNA sequence, and often only shorter length’s of replication may be possible. Some new polymerases, such as “Phusion”, are now available which are claimed to have both high fidelity and the capablity to replicate sequences of > 18 kb. It is certainly possible with care to obtain intact bacterial genomes and bacteriophage genomes, and it is not generally necessary to cut these with a restriction endonuclease before PCR. Eukaryotic genomes with multiple chromosomes are a different matter. It is possible to purify the intact DNA molecules of small eukaryotic chromosomes using various pulsed field electrophoretic techniques (http://www.nal.usda.gov/pgdic/Probe/v2n3/puls.html). However, for routine PCR studies this is not generally necessary, and DNA can be extracted and purified in a manner that yields fragmented (but large sized) DNA.
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