Copy Number Applications

Description

Novallele Copy
Number Assays

The Novallele copy number assays provide a simple method to investigate deletions and duplications of individual genes. By using high-resolution melting (HRM) to analyze relative fluorescence, users can quickly assign copy numbers to all of their samples. Each assay delivers accurate and reproducible copy number calls without requiring specific instrumentation – you just need an HRM-compatible thermocycler.

 

Determining Copy Number using HRM

To determine copy number, the Novallele copy number assays compare the relative fluorescence values of samples to the fluorescence of characterized controls for the target of interest. Analyzing the relative fluorescence of samples is a simple, fast, and sensitive method to determine copy number.

Comparing the relative fluorescence across a pool of samples requires stopping the reactions prior to reaching the fluorescence plateau. The cycle number is less than traditional PCR protocols, and the chemistry is optimized to keep the amplification in the linear phase until the samples are melted.

 

To generate the relative fluorescence data required, the gene of interest along with an internal reference that is conserved at 2 copies are both amplified and their fluorescence captured by HRM. Using deoxynucleotide triphosphates (dNTPs) as the limiting reagent in the PCR, the gene of interest and the internal reference compete against one another in the reaction mixture. Samples with duplications in the gene of interest will preferentially amplify over the internal reference since the gene of interest has more primer binding sites; inversely, the internal reference will preferentially amplify over samples with deletions in the gene of interest since the internal reference has more primer binding sites. In order to keep the fluorescence values relative between the amplified products, PCR is stopped during the linear amplification stage. This is accomplished by reducing the number of PCR cycles to 25.

A derivative plot generated using the SMN1 Novallele Copy Number Assay. The 0-copy curves are black; the 1-copy curves are blue; the 2-copy curves are red; the 3-copy curves are green. The lower temperature melt curves on the left are those of the gene of interest, in this case SMN1. The higher temperature melt curves on the right are those of the internal reference.

 

A difference plot generated using the SMN1 Novallele Copy Number Assay. The 0-copy curves are black; the 1-copy curves are blue; the 2-copy curves are red; the 3-copy curves are green.

 

To analyze the data, the Novallele HRM Analyzer software normalizes the HRM data for background fluorescence and corrects for temperature shifts. The software groups samples with characterized copy number controls. Characterized controls are required for each run as the copy number for each sample is assigned based on the clustering of the sample with a characterized control. 

 

 

A Simple Workflow

The Novallele copy number assays use a fast and simple PCR-based workflow. Any HRM-capable thermocycler can run the Novallele copy number assays, and some thermocyclers are fast enough to provide results in under an hour. The Novallele copy number assays and Novallele Copy Number Mastermix are optimized to reduce data variability between HRM-compatible thermocyclers or different DNA extraction protocols. This optimization, coupled with easy visual data analysis, generates quality data without requiring replicate samples to make a copy number determination.

Get Answers Faster

 

Simplify your SMN1 and SMN2 copy number analysis and quickly generate robust, reproducible copy number data.

 

  • Accurate — functionally tested assays
  • Versatile — use any HRM-capable thermocycler
  • Fast — results in under an hour
  • Easy — no replicates required
 

Order Now

Product

Catalog Number

Number of Reactions (10 uL)

 
Novallele Copy Number Mastermix 40797 200 View Details
SMN1 Novallele Copy Number Assay 40798 200 View Details
SMN2 Novallele Copy Number Assay 40799 200 View Details

Resources

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