Whole Genome Amplification - (Dec/07/2009 )
I'm looking to do a ChIP-on-chip. I've read over the various methods for amplifying your enriched/IP'd DNA. I was going to settle on LMPCR as this seems to be the most common. But I came across a write-up describing another method that looks a LOT simpler and doesn't depend on blunting and end ligation. A mentor/prof suggested it to me, and so I printed it up.
Moqtaderi Z, Struhl K. Defining in vivo targets of nuclear proteins by chromatin immunoprecipitation and microarray analysis. Curr Protoc Mol Biol. 2004 Nov;Chapter 21:Unit 21.9
It's for Yeast ChIP, but there doesn't appear to be any reason it can't be adapted for mammalian cells.
Basically, you use two primers, one for each of two PCR reactions. The first is a 5' tagged random octamer. i.e. (M13FWDseq)NNNNNNNN, and the other (in this case) would be M13.
You amplify, 2 cycles, with the random octamer and sequenase, leaving a bunch of fragments of random size tagged at either end with M13FWD. Then you purify and do a typical amplification with primers that match the 5' tag - M13FWD in this example case, amino-allyl dNTP, and Taq pol, for 35 cycles. Conjugate dyes and hybridize.
This seems much much easier and less delicate than the LMPCR. Everywhere I've read tells me the LMPCR can be finicky for unknown reasons, and you should expect it to fail the first, second, and possibly even the third time you attempt it.
So my question is, why isn't anyone using this method? There has to be a reason. I've combed the web, and I can't find anyone else who uses it, warns about it, or has even mentioned it. Does anyone have any experience with this method?
Does anyone have any thoughts or experience with this protocol?
MunkySpunk on Dec 7 2009, 01:53 PM said:
There are 3 commonly used protocols for CHIP-on-chip amplification: (1) random PCR, (2) LM-PCR, (3) WGA. The protocol you mentioned above is random (amplification) PCR. Affymetrix provides detail protocol on their website http://www.affymetrix.com/products_service...omoter.affx#1_4 . Just click “Tool & Data” button and download manual “chromatin Immunoprecipitation Assay Protocol”. You have to register it first. It’s free.
Additional website for random PCR protocol (or similar protocol):
• Methods. 2007 Mar;41(3):304-11. “Genome-wide analysis of chromatin status using tiling microarrays.” Shivaswamy S, Iyer VR.
• Nat Methods. 2005 Mar;2(3):219-24. “Profiling DNA methylation patterns using genomic tiling microarrays.” Lippman Z, Gendrel AV, Colot V, Martienssen R.
• UHN Microarray Center http://www.microarrays.ca/info/protocols.html Chromatin Immunoprecipitation Protocol for Microarray Analysis – Staph A Cell Method or bead Method
People use this method. There are published papers by using random PCR amplification with mammalian cells line for CHIP-on-chip.
Other protocol provides random PCR (same as Affy) and WGA amplification methods.
Methods Mol Biol. 2009;523:341-66. “ChIP on chip assays: genome-wide analysis of transcription factor binding and histone modifications.” Pillai S, Chellappan SP.
Paper compares LM-PCR and WGA :
Biotechniques. 2006 Nov;41(5):577-80. “Comparison of sample preparation methods for ChIP-chip assays.” O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ.
Just try to comb web harder and have fun.