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High-Fidelity PCR with a Novel Polymerase Mixture

an successfully amplify GC-rich templates requiring denaturing temperatures of 96-98C.11 A limitation of Pfu DNA polymerase is its relatively slow polymerization rate, incorporating approximately 550 nucleotides per minute.2 However, when used with a minimum extension time of 2 minutes per kb,12 this enzyme can successfully synthesize PCR products up to 12 kb in cloned Pfu reaction buffer13 and up to 25 kb in Taq reaction buffer (table 1).

PCR Product Length (kb)

Extension time required

Error Rates 1,2

Vector Target Yield3 Genomic Target Yield3 Min/kb target (x10-6)

(high)

(low)

(high)

(low) Pfu DNA polymerase

2+4

1.3

0-5
'"/>

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