whole genomic amplification - (Feb/18/2008 )
has anybody ever used a whole genomic amplification kit? my boss wants us to use it to amplify DNA from single cells and test it with CGH. We have to chose which one to buy (I found several: ampliQ, genomiphi, repli-G, and genomePlex) to test it on human lymphocytes. Any advice welcome!
No, haven't tried that.
Which is mostly used in published papers?
well, I have found very few papers...and it seems like every paper that describes the usefullness of one of this kits...belongs to the group that produced the kit....even one that compares the four of them: "whole genome amplification on DNA filter paper blod spot samples: an evaluation of selected systems, 2007,genetic testing, Soresen"...
I tried both Genomiphi and Repli-G .
Genomiphi is quite good for daily use (PCRs....). I have amplified DNA from more than 100 individuals and make PCR on it.
It s chiper product and faster protocol (2h with the new one) compare to Repli-G
Repli-G give better yield, and fragments have longer size than for genomifi. I will recommand it for CGH for this reason....
I did not try the others.
But I have other question, why do you need to amplify your DNA, you should have enough DNA from cell culture extraction? As far as I remember, 1 µg of DNA are necessary for hybridization..then with several culture plates....
thanks, The reason for single cells, I think, is that we have had unculturable amniocytes or with moulds (even if rarely) and could not do caryotyping....but very few cells were still alive....so we could have done aCGH....so I think lynphocytes are just a test...and more affordable...and then we'll try amniocites.
At the end I am testing repli-g.....and I had nice amplification, but the problem now is that I have to check if I have amplified all the Chromosomes and the same quantity for both alleles for heterozygos traits...so it could be possible that I will ask you some more question in the future if u do not mind! : )!!
Hope your WGA is going well! My lab has used REPLi-g in the past (before my time), but switched to something called IntensiPhi. Not sure if there was another reason besides cost...anyway, I have used the IntensiPhi protocol to amplify low-yield human DNA samples from buccal swabs and have performed PCR-based genotyping of length polymorphisms. While differential amplification of heterozygous alleles is a concern, I haven't an easy way to check. I know other people here have performed the TaqMan SNP assays on amplified samples; their call rates are lower than with the unamplified samples, I believe.
thanx, at the end we decided to use microsatellites to check for differential amplification which sounded better than using FISH (our first idea)....we had good amplification for many of them..but still several allele drop out as I supposed, so it needs lots of working in varying PCR conditions etc....the problem is that for money reason I could only try one sample....so I am waiting to know from my boss how much money he is willing to invest in the project before moving on....in the mt I will check the other kit you suggested : ) !!!