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Last Login: Wednesday, November 21, 2007 9:18 PM
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| We are using the Affymetrix amplification protocol to prepare samples for hybridization. This is a 2-step PCR protocol using 15 cycles per step, and starting with approximately 5 ng IP-DNA (quantified by nano-drop). The amplification also includes incorporation of dUTP in the PCR product to facilitate later steps of DNA fragmentation and labeling prior to hybridization. Typically we end up with 1-5 ug DNA following the PCR protocol. IP-DNA and mockIP-DNA (no antibody) are used as starting templates. Enrichment of the IP-DNA is confirmed by amplification of a known target prior to the PCR protocol. However, on several occasions we have observed loss of this specific enrichment after the PCR protocol. We have ruled out the possibility that a proof-reading Taq polymerase was used in the analysis of the post-PCR material. We are currently testing other whole genome amplification strategies, eg Sigma GenomePlex to see if this improves the situation. We are also running PCR test where we omit the dUTP. However, if these alternative PCR methods are successful, we would then need to use DNaseI digestion for fragmentation of the PCR products. Another possibility is to reduce the number of PCR cycles in the Affymetrix protocol, but it is difficult to estimate how many cycles are needed to provide sufficient material for hybridization. Has anyone else experienced loss of specific enrichment during PCR of the IP-DNA, and/or have any suggestions regarding this issue? We do not have a great deal of experience with the ChIP-chip assay, and any comments would be welcome.
Geoff Neale
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| Hi Geoff, We also see the same thing here, although it happens not that often anymore . I changed the second pcr amplification and made it into one pcr program with 30 cycles. In almost all cases I still have the enrichement after this pcr step. Sometimes we have to lower the cycles in the second pcr, but this is something you will just have to try. You could also do Linker mediated pcr first and then do the second pcr with dUTP in there so you can use udg/ape1, for some samples this seems to work better. cheers, Jos
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Last Login: Thursday, November 08, 2007 2:48 AM
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hello, we also observe the same bias after the amplification of enriched DNA. Now, i think some factors may be helpful to reduce this happen,
1)reduce the cycles of PCR amplification ,such as 25 cycles ,through several array experiments, we found less cycles may be better for obtaining more binding sites, replication of biological may be reasonable than amplification of more cycles.
2)through ChIP-qPCR to verify some specific genes, you can control or evaluate the suitable cycles. 
ChIP-chip
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Last Login: Monday, January 28, 2008 1:56 PM
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Dear all,
I also see an increased PCR bias when I increase the cycle number. I found 24 cycles to work well for amplification of 5 ng. Using a different taq or simply more taq might also help.
I use 6 different controls (not enriched loci) to check for PCR bias after amplifiation. For these controls I calculate the delta ct values of 2ng Amplified total input versus 2ng not amplified total input. Due to the PCR-bias, I see ratios of up to 10 fold after amplification. - To get this far, I allready spend a lot of time in optimising PCR conditions. First, I saw ratios of up to 128 - much higher then the enrichment in my IP samples.
! I would be very interested what the highest ratios between controls are that you see after amplification. !
Thanks,
Fridtjof
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