Detecting Copy Number Variation (CNV) with Digital PCR - Ask TaqMan #28

Submit your question: http://bit.ly/1cgFftk
Copy Number Variations, commonly referred to as CNV's, can result in having either too many or too few of a particular loci. Like other types of genetic variation, some CNVs may be responsible for a substantial amount of human phenotypic variability, complex behavioral traits and susceptibility or resistance to disease.

When performing CNV with digital PCR, you're performing an absolute quantitation of the actual locus of interest. To convert the absolute quantities to a copy number, you want to compare your target to a reference locus that has not been amplified or changed in any way. Typically the reference will be diploid, but it doesn't have to be. It can be anything, as long as the number of copies is known beforehand.

For copy number variation determination with digital PCR, you will need two assays, one that is specific for your target locus and the other that is specific for the normal diploid locus. When quantifying, you will take the ratio of absolute quantities of the target locus to the absolute quantities of the reference locus. Depending on the target, a ratio of more than 1 could suggest an amplification, and a ratio of less than one suggests a deletion. Sometimes your reference itself can have a copy number change. This will alter a ratio in such a way that makes the data unclear. Especially in the case of cancer samples, it is a good idea to use two references.

LOVORKA:

Let's focus on the sample preparation in more detail. To achieve an accurate copy number for your target locus, you have to consider the neighborhood in which your locus is found. If the locus is suspected to be a tandem duplication, the use of a restriction enzyme is very important. But why is this so important for digital PCR?

Let's look at a scenario where we would like to measure tandem copies. If we were to proceed with without the use of a restriction enzyme the tandem copies will both occupy the same through hole of the chip thereby giving you an underestimated copy number.

Видео Detecting Copy Number Variation (CNV) with Digital PCR - Ask TaqMan #28 канала Thermo Fisher Scientific