Enhancing PCR amplification and sequencing using DNA-binding proteins.
The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.
Rapley
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Div. of Biological Sciences, School of Natural and Environmental Sciences, Coventry University, UK.
D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D016133:Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D011938:Rec A Recombinases; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase