The Golden Gate assay can be used to efficiently detect, confirm and genotype most forms of Copy Number Variation (also called CNV or structural variation) greater than ~20bp in size, assuming that the region being assayed contains some non-repetitive DNA.

Copy Number Variation consists of the increase or decrease of genomic sequence in a genome, and is usually considered to be sequence lengths greater than a single nucleotide or small two or three nucleotide variants (SNPs and in/dels, respectively). Most catalogued CNV in the human genome is greater than 5000 basepairs, but this is likely due to an ascertainment bias as there is evidence to suggest that much more CNV exists that is between 3 and 5000 bp.

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How Does This Work?

The Basics

The process of detecting CNVs relies on the same Golden Gate technology used to genotype SNPs, but differs analysis of the data.

As in all Golden Gate assays, sequence-specific oligo probes are designed for up to 384 loci of interest per multiplexed reaction. For CNVs it is not important that the probes flank a specific SNP, although the presence of such a SNP will not affect the assay. Typically, if the existence of the CNV is speculative, the probes need to span the region of interest, with a minimum of 7 probes placed to increase the detection odds.

The oligos are used to PCR amplify and fluorescently label the regions.

Then, these amplification products are hybridized to and immobilized on a special substrate built by Illumina, called a VeraCode bead. From here, the Illumina BeadXpress machine can automatically determine the identity of the oligo primer hybridized to the VeraCode beads, and measure and quantify the fluorescent signal (thus indicating the genotype) for each amplified probe region.

The presence, absence and intensity of the different fluorescent dyes bound to each probe indicates the presence and absence and extent of Copy Number Variation in the regions of interest.

For a more detailed description of the mechanics and chemistry, we recommend that you consult the Illumina web site.

How to Get Started

For CNV assays, we strongly recommend that our clients call us for recommendations and assistance from our highly experienced team, before generating an oligo list.

Once an oligo list has been created for the loci of interest, this file is provided to Illumina as one part of the order. Illumina will use the SNP and oligo list to create an OPA (oligo pool array) which is actually the list of which VeraCode beads will match to which oligos.

Illumina then sends the OPA and associated reagents to our laboratory. Once you’ve sent your DNA, we initiate the standard Golden Gate assay using your OPA. When your data is generated and analyzed for accuracy, we send it to you.