8. GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects
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The amplification of GC-rich templates by any PCR method is usually a difficult task and despite the development of modified
methods and conditions, this type of amplification still remains a specific case approach. Problems usually observed with
GC-rich DNA are constraint of template amplification by stable secondary structures that stall or reduce the DNA polymerase
progress, and the presence of secondary annealing sites giving rise to nonspecific amplified bands. This latter point is not
exclusive to GC-rich templates but is frequently encountered in other types of templates. In order to design a more general
method for GC-rich templates, different DNA polymerases were compared in combination with different organic solvents with
the purpose of abolishing stable secondary structures (1). Our attention focused on the inverse polymerase chain reaction (iPCR) used to perform site-directed mutagenesis (1
2). This very attractive method req⪚res a single pair of primers and involves the amplification of the whole recombinant plasmid,
a difficult step with high GC-content DNA. Inverse PCR also proves useful in cloning missing parts of genes by using a self-ligated
genomic DNA fragment as template.
Affiliation(s): (2) Sainte-Justine Hospital Research Center, Montreal, Canada
(3) Centre D’Ingénierie des Protéines, Université de Liége, Liege, Belgium
(3) Centre D’Ingénierie des Protéines, Université de Liége, Liege, Belgium
Book Title: PCR Cloning Protocols
Series: Methods in Molecular Biology | Volume: 192 | Pub. Date: Apr-01-2002 | Page Range: 75-80 | DOI: 10.1385/1-59259-177-9:075
Subject: Genetics/Genomics
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