Can you tell me how to determine gene copy number without using WGS or Southern blotting? Any thoughts? I's told by a colleague to use array CGH. This is kind of like a microarray designed against specific regions of the genome. You look at the signal generated from a universal pooled reference DNA versus the sample of interest. If the signal for your sample is greater than the reference that means there is a copy number gain, if the value for the sample is less than the reference, that means that you have a loss. However, how can you detect hybrid CNVs?
Dear Shengwen,
Another way of looking for CNVs could using real time PCR. For this, the region of interest needs to be known. As a validation procedure, this might be a good approach comparing a sample of interest vs controls.
Best
Gilles
aCGH is a gold standard techniques for CNV analysis, but there is also a newer technology by NanoString (see http://www.nanostring.com/applications/CNV).
There are different ways CNVs can be detected or validated, but the method you choose will depend on what you are trying to find.
- If you want to detect CNVs in multiple genes or regions of a genome (other than by WGS) you can use arrayCGH (aCGH, as mentioned above) or by SNP arrays such as (Affymetrix SNP 6.0 or CytoScanHD)
- If you want to detect CNV in a specific gene then real time PCR (qPCR, as mentioned above) or digital droplet PCR (ddPCR) approach could be used.
However, all of these techniques look at the average copy number in a pool of cells from which one extracted DNA (unless you do single cell WGS). Some CNV events may be clonal (I'm not sure if this is what you mean by hybrid CNVs). If you want to assess the clonality of a CNV in a given sample you can use methods such as fluorescence in situ hybridization (FISH). FISH has the some advantages over the other methods as a validation tool because if you had a sample where 1/10 cells had 4 copies of a gene and 9/10 had 2 copies of that same gene, arrayCGH, SNP arrays, WGS, qPCR and ddPCR will all report that the sample has 2.2 copies, which might be interpreted as "normal copy number" whereas in fact there is a significant CNV but in a sub-population of the cells.
Cheers.
Multiplex ligation dependent probe amplification (MLPA). Look at MLPA.com
i suggest you to use DDPCR, Digital PCR, an end-point PCR method, does not rely on assumptions of perfect amplification efficiency or on reference standards to reach high precision in copy number enumeration
for the protocol and how to use you can read this protocol
Chapter Copy Number Variation Analysis by Droplet Digital PCR
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