Key components of Genesky’s proprietary AccuCopy® assay method (Patent ID: 201010180551.2) involves a set of locus-specific amplification primer pairs, a set of competitive DNA fragments and a delicately optimized multiplex-PCR reaction system. AccuCopy® method is illustrated in figure 1. Generally, single-copy gene (that is, two copies in a diploid genome) is selected as reference. Long competitive double-strand DNA sequence is synthesized for each target and reference genomic segment. Each competitive DNA sequence (C) is highly similar to its human homology in sample DNAs (S), while they are different in length for several base pairs (bp). The red bars in this figure indicate the oligonucleotide sequences in human homologies that are deleted in their competitive DNA sequences. The black bar indicates the 5’ flanking sequence of the deleted part and the gray bar indicates its 3’ flanking sequence. Both of these flanking sequences are identical between human homology and its competitive DNA. The synthesized competitive DNAs for target and reference segments are first mixed with a defined amount of genomic DNA of tested sample. Multiplex fluorescence competitive PCR is performed to simultaneously amplify all reference and target segments from both sample DNA and competitive DNA. The blue dots indicate a blue fluorescent group labeled to each forward primer. Amplicons with different sizes are separated by capillary electrophoresis and the peak ratio of sample DNA to competitive DNA (S/C) for each segment is calculated. After normalization by reference segment’s peak ratio, the copy number ratio of each target segment to reference can be easily determined by its peak ratios divided by reference’s peak ratio, when the competitive DNA segments are well balanced in molecular number in the DNA mixture. The copy number of target segment can be identified, given that the copy number of reference segment is known.
Technique Highlights
– Multiplexing Capability Allowing simultaneous CNV detection at 12-16 genomic loci in a single reaction (including 8-
12 target loci and 3-4 reference loci).
– High Accuracy At least three reference genes used; measurement CV < 5% on average.
– High Sensitivity CNV loci with up to 6 copies can be accurately quantified.
– High Throughput Requiring less than 4 hours to rapidly and accurately determine CNV in large scale of samples.
– Low Cost Requiring few replicates to obtain highly reliable results, thus highly cost-effective.
– High Flexibility According to customer’s specific project need, we can help design and synthesize custom sequence
panels and develop custom ready-to-use testing kit. Customers only need a florescent capillary electrophoresis
analyzer to conduct highly efficient CNV multiplex tests with any qualified samples on their own bench.
Sample Requirement
Sample Form: Genomic DNA
Sample Quantity: >= 5ug
Sample Conc.: >= 100ng/ul
Sample Purity: OD260/280 = 1.7 ~ 2.0
Sample without severe degradation
Service Workflow
1. Project strategy consultation and custom experimental design
2. Sample receiving
3. DNA extraction and QC
4. AccuCopy assay
5. Project final report
Deliverables
1. CNV analysis result2. QC result
Reference
Du, R., et al., Efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification. J Hum Genet, 2012. 57(8): p. 545-51.
