Critical factors that influence downstream PCR-based analysis following MDA

Detection of a specific genetic loci from a paraffin-embedded tissue sample using PCR is strongly influenced by the following factors:

• Copy number
• The degree of cross-linking
• Amplicon size

Copy number: The larger the amount of DNA, and therefore the copy number of the genome, the more likely a specific locus will be detected by PCR after whole genome amplification. However, quantification of DNA from paraffin-embedded samples can be challenging due to the varying amount of contaminating substances isolated with the DNA. Contamination can be identified using a UV scan. In contrast, using single A₂₆₀ measurements instead of a UV scan (220 to 320 nm) will lead to an overestimation of DNA concentration. By overestimating the amount of input DNA in PCR or other downstream experiments, low performance may be mistakenly observed.

Cross-links: The higher degree of crosslinking in a DNA sample, the lower the performance of an amplification reaction such as whole genome amplification.

Amplicon size: The smaller the size of the amplicon used for PCR-based analysis of FFPE DNA, the greater the chance of detecting a specific locus. Real-time PCR of intact DNA compared with DNA from paraffin-embedded samples shows that increasing amplicon sizes dramatically reduces the number of detectable genome equivalents.