Quantitative PCR (qPCR) has been used for analyzing gene expression and quantifying copy number variation. qPCR expression analysis includes amplifying a reference locus with a known copy number and a test locus with an unknown copy number. Today, copy number qPCR analysis has been employed in multiple diseases, such as determining SMN1 and SMN2 copy numbers in spinal muscular atrophy and determining her2/neu amplification for human breast carcinoma. 

Copy number analysis using qPCR assays involves two approaches: intercalating dyes and fluorescent eyes. In every cycle of qPCR assays, the fluorescence doubles, and the starting template can be determined from the cycles required to reach a prespecified fluorescence threshold. Post-sample preparation, the qPCR experiment can take half a day. The current article focuses on the science of qPCR copy number variation assay. However, similar to LC-MS drug testing, qPCR requires adequate development and validation efforts. 

qPCR copy number analysis

To measure DNA copy number with qPCR assays, the sequences should be unique to that gene, and the amplicon should be present within the axon or intron. Besides, it should include two copies of a control gene.

qPCR is a modification of conventional PCR that can quantify the target DNA using intercalating or fluorescent dyes. RT-qPCR can identify these PCR products in real-time. qPCR can provide relative or absolute quantification of DNA, including mitochondrial DNA, cyclic DNA, and chromosomal DNA. qPCR assays have multiple applications in diagnosing infectious agents or characterizing genetic abnormalities. Robust qPCR assay can evaluate copy number analysis. However, the assay and primers require adequate optimization for robust and reproducible results. Let us now study some critical parameters of qPCR assays. 

Primer design

• Choose regions with higher GC content and avoid the ones with long repeated single bases.
• Avoid copy number polymorphism and single nucleated polymorphism at the site of annealing.
• Short PCR products are perfect as they are amplified at a higher efficiency, and hence, primers with 75 to 200 base pairs amplification products are ideal for qPCR assays.

Probe design

• Scientists can combine primers with DNA-binding dyes to monitor double-standard DNA. Fluorescently labeled probes can provide greater specificity. 
• Keep the probes less than 13 nucleotides.
• Maintain the melting temperature of the hydrolysis probe up to 5 to 10 degrees Celsius higher than primers.

Reaction efficiency

Scientists can construct a standard curve to determine the reaction efficiency of qPCR assays. A standard curve can determine reproducibility, linear dynamic range, and efficiency. Researchers can produce standard curves for genes of interest and internal control genes by plotting the log of each known concentration against the CT value for that specific concentration. 

Verifying assay specificity

Scientists perform melting curve analysis to verify assay specificity. Primer dimers and non-specific products can reduce the accuracy and amplification efficiency. Hence, it is critical to verify assay specificity as non-specific amplification can produce products melting at temperatures above and below the desired product and primer melting at lower temperatures than the desired product. In conclusion, qPCR assays are a simple yet effective technique to quantify DNA copy numbers in human samples.