Towards the absolute quantification of DNA by PCR

Amplification techniques such as the Polymerase Chain Reaction (PCR) are held to be largely qualitative procedures and are widely used as such. Since the efficiency of amplification is less than perfect, small changes in efficiency can yield dramatic differences in the final amount of product generated. Despite this unpredictability the exquisite sensitivity of PCR makes the demanding goal of absolute quantification highly desirable. Consequently, the use of this technique for the quantification of nucleic acids has increased at an exponential rate. However, the ability of PCR to accurately quantify absolute levels of DNA is still not universally accepted. The overall aim of this investigation was to determine the critical factors affecting the quantification of DNA using PCR and to use these findings to develop an assay for the absolute quantification of DNA in a model system. The novel work presented here illustrates the need for careful examination of sequences for GC-rich domains which could give rise to stable secondary structures and reduce the efficiency of amplification by serving as termination sites. To determine the accuracy of competitive PCR, CE and IP-RP-HPLC were employed to quantify PCR- products. These two techniques provided valuable information on the identification and elimination of sources of error which led to improvements in speed, accuracy and precision, as well as ease of quantification by PCR. They also yielded information on the process of heteroduplex formation whilst simultaneously revealing assay limitations. Consequently, the on-line fluorescence monitoring of PCR was used as an alternative method for the quantification of Legionella pneumophila. This technique was highly reproducible however, mispriming and the subsequent amplification of non-specific PCR products limited the level of detection. The Y-end labelling of degraded DNA with DIG prevented short DNA fragments from mispriming (and consequently extending) allowing the amplification of DNA targets. Therefore, to reduce mispriming and hence improve assay sensitivity, this approach was adapted for the first time to produce 5'-degenerate, 3'- DIG-terminated competitive primer analogues. These analogues, coupled with the use of the LightcyclerTM, allowed the detection and absolute quantification of a single cell of Legionella pneumophila. This is the first time that this level of sensitivity has been achieved using this type of assay. This technique should provide a very rapid and sensitive alternative for quantification compared to the other, more expensive technologies available at present