Wilhelm et al. 2003

Wilhelm J, Pingoud A, Hahn M. 2003. Real-time PCR-based method for the estimation of genome sizes. Nucleic Acids Research 31: e56.

These authors describe and demonstrate a new method for genome size estimation, based on real-time PCR (RT-PCR). The RT-PCR process is used to count copies of a target sequence in a sample of genomic DNA of unknown genome size, simultaneously with the amplification of the same sequence in a standard DNA present in the same reaction tube. As with other methods of genome size estimation, the known standard is used to calculate the unknown genome size, though the actual calculation is different from that employed in flow cytometry or Feulgen image analysis densitometry.

Estimation by RT-PCR involves calculation of the unknown genome size by dividing the total mass of genomic DNA by the number of copies of the target sequence as detected by fluorescence during the amplification reactions. Typically, a target sequence that is known to exist one-copy-per-genome is used. The co-prepared standard is used to calibrate fluorescence intensity and copy number.

This method only works if two conditions can be met: a) the absolute copy number of the target sequence in the standard is known and b) the absolute mass concentration of the genomic DNA in the sample can be determined accurately. Both conditions can be met using the absorbance of the sample at 260 nm (UV-260 abs). Protein contamination will prevent accurate readings, and can be detected by determining the absorbance properties of the sample at the full range of wavelengths between 260 and 320 nm. RNA will also interfere with measurements, at it absorbs similarly to DNA at UV wavelengths; it should be removed with an exhaustive treatment of RNAase.

The copy-concentration of the standard can be determined from pure samples by dividing the measured mass of standard DNA by the known fragment size. To ensure accurate calibration, the standard’s sequence should be identical to the target sequence amplified from the unknown; thus the ideal standard is a fragment that contains the RT-PCR “inner primer” sites and is of known fragment length. This can be determined by gel electrophoresis if the primer positions are not already reliably known.

These authors conclude that RT-PCR genome size estimation is accurate, precise, and useful in situations where other techniques are impractical. For example, they state that it can be used on samples that cannot be used in flow cytometry. Though they provide no examples of such situations, I can think of at least one widespread condition: that of specimens preserved in ethanol with destroyed or damaged nuclei but undamaged DNA. These authors make no mention of their method’s relative advantages compared to Feulgen-based methods.