Sensitivity of the Analyses

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Considerations used in determining the sensitivity of our analysis:

These reports describe the CSGE method in detection of 68 polymorphisms or mutations in various genes. Based on the results, authors suggested that CSGE can detect almost all changes if the primers are designed in a manner that leaves >50bp "buffer sequence" in both 5' and 3' ends of the fragment. Also, isolated GC-rich regions were noted to be difficult areas for the detection of single-base pair sequence variations. According to these articles the sensitivity of the CSGE in detecting single base-pair mutations was either 95% (with the 50bp end-sequence effect considered i.e. five sequence variations less than 50bp from the end of the sequence were excluded) or 88% (all changes).

Korkko J, Annunen S, Prockop DJ & Ala-Kokko L. Conformation sensitive gel electrophoresis (CSGE) for simple and accurate detection of mutations. Comparison with denaturing gradient gel electrophoresis (DGGE) and sequencing. Proc Natl Acad SCI USA, in press.

This article describes experiences and findings gathered by us during the two-year process of testing if CSGE could be used for scanning PCR products prior to nucleotide sequencing without sacrificing the sensitivity of mutation detection. By utilizing known sequence variations and their detection by CSGE we suggested new conditions for CSGE analysis. By employing these conditions we were able to detect virtually all 76 sequence variations tested. Importantly, these changes included the three changes in high melting regions that were not detected in previous articles. Therefore, we suggest that CSGE can detect virtually all single base-pair changes (~99%).

Large deletions and insertions may go undetected as well as homozygous mutations. Combined, these mutation types account for 2-3% of reported mutations in genes for type I and II collagens.

Additional factors that possibly could decrease the sensitivity:
        Allele-specific inhibition of the PCR amplification due to mutation (secondary structures?)
        Rare polymorphisms in the primer regions

However, these factors are mostly hypothetical and therefore, it is very difficult to estimate their effect, if there is any, to the sensitivity.

In summary, we feel confident to say that 
our analysis can detect more than 90% of the mutations 
in the genes analyzed.