I have a problem with DNA extracted from FFPE samples (human heart). Our method for mtDNA amplification and sequencing (twelve fragments) that has been working perfectly works now with few samples only. I have optimized PCR and the samples have been purified with commercial kit (Illumina). The purification resulted in weak improvement in the amplification. I will test WGA kits to get more mtDNA next.
I know that I could amplify smaller fragments but the sequencing gets too expensive with small fragments. Furthermore, I do not have the heart tissue, only DNA.
So, have you been struggling with FFPE samples? Have you solved the problem with mtDNA fragmentation?
WBR
Laura
I need to amplify much larger amplicons, from 1000 to 2500 bp. Shorter fragments already work. I have now tried the first WGA kit (Repli-g FFPE WGA kit) and it did not work. I was hoping that there is enough intact mtDNA to get it amplified, but it seems that mtDNA is too fragmented. Next, I will test another WGA kit. After that, it is time to try 3-pieces PCR and build the genome.
I am following ResearchGate on a daily basis. So, advices still needed!
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