Relative Amplification Efficiency of Differently Sized Templates by Long-Distance PCR

Relative amplification efficiency of differently sized templates by long-distance PCR.

Multiplex co-amplification of 24 retinoblastoma gene exons after pre-amplification by long-distance PCR.

Polymerase chain reaction (PCR) amplification has become the method of choice for preparing the DNA template in mutation analysis from complex mixtures of DNA or RNA molecules. This strategy is optimal for small genes or genes with mutational hotspots. However, PCR-based mutation detection is neither labour nor cost effective when large or multiple genes, with many target fragments, are involve...

Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates.

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III per...

Improved PCR Amplification of Broad Spectrum GC DNA Templates

Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate ...

Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method.

Polymerase stop assays used to quantify DNA damage assume that single lesions are sufficient to block polymerase progression. To test the effect of specific lesions on PCR amplification efficiency, we amplified synthetic 90 base oligonucleotides containing normal or modified DNA bases using real-time PCR and determined the relative threshold cycle amplification efficiency of each template. We f...