CNVplex® is a multiplex ligation-dependent probe amplification method and key steps of the technique are illustrated in Figure1. Each CNVplex reaction system contains several pairs of probes, with the left probe consisting of a 5’ universal amplification primer followed by a locus-complementary left sequence, the right probe consisting of a locus-complementary right sequence followed by a linker sequence and a 3’ universal amplification primer. The length of the linker sequence can be adjusted in order to discriminate the copy number of multiple loci in capillary electrophoresis. CNVplex® determines copy number of targeted locus by the hybridization of two locus-complementary probes to the targeted locus. The amount of probes hybridized to the locus is proportional to the copy number of locus. Two probes are positioned next to each other upon hybridization, and then are jointed together by the ligase. Only probes with a perfect match can be ligated. Then PCR amplification is performed using primers with universal sequence. Other than adjusting the length of the linker sequence, the though-put of CNVplex® can be further increased by using four different florescent labelling for PCR amplification as well as the introduction of lengthening double-ligation system. Amplification products are separated by size and colour using capillary electrophoresis and the relative amount of PCR product is proportional to the original copy number of the target loci.
Technique Highlights
– High Throughput Simultaneous quantification of copy number at 48 - 480 loci, significantly increase CNV detection
efficiency.
– High Accuracy Highly specific ligation based method. Amplification of ligation products using universal primers
makes amplification efficiency highly similar, average detection CV < 10%.
– High Resolution Accurately quantify loci within 6 copies.
– High Reproducibility Provide highly stable detection result, help you save cost by reducing unnecessary
experimental replicates.
– High Efficiency and Fast Turnaround Time Utilize short probes that can be directly synthesized without cloning,
thus significantly streamline and accelerate the procedure.
Sample Requirement
Sample Form: Genomic DNA
Sample Conc.: >=100ng/ul
Sample Quantity: OD260/280 = 1.7~2.0
Sample without severe degradation
Service Workflow
1. Custom sequencing experimental design2. Sample receiving
3. DNA extraction and QC
4. CNVplex assay
5. Project final report
Deliverables
1. CNV analysis result
2. QC result
Reference
Zhang, X., et al., A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease. BMC Genomics, 2015. 16: p. 364.
