qPCR For Deletion Analysis: A Practical Guide

Quantitative PCR (qPCR) isn’t just for measuring gene expression; it’s also a powerful tool for detecting gene deletions, especially when you suspect a heterozygous deletion in a particular gene region. In this article, we’ll walk you through how to use SYBR Green-based qPCR to validate deletions, interpret results using the ΔCt and ΔΔCt method, and avoid common pitfalls like polymorphic dropout.

Why Use qPCR for Deletion Analysis?

Techniques like MLPA, array CGH, or exome CNV calling are the gold standard for copy number changes, but they may not always be available or feasible. qPCR can act as a quick, cost-effective confirmation method if you design it well and interpret it carefully.

You’re not detecting alleles, you’re detecting how much DNA is present for the region you’re targeting. Therefore, if there’s a deletion, you’ll receive a lower signal (higher Ct).

To understand the basic steps of qPCR, you can go through this article:

What Kind of Deletion Can You Detect?

• Heterozygous deletions (1 copy) → ~50% signal (0.5x)
• Homozygous deletions (0 copies) → No amplification (or very high Ct)
• Not suitable for: very small deletions (<20–30 bp), intronic deletions without proper primer design, or low-level mosaicism.

Step-by-Step Protocol

1. Primer Design

• Target the exon or region suspected to be deleted.
• Amplicon size: 80–150 bp
• Avoid SNPs or polymorphic regions in primer binding sites (use Ensembl/UCSC)
• Also design primers for a reference gene (e.g., GAPDH, RNase P, ACTB)

2. DNA Samples

• Test sample (patient)
• Control sample (normal DNA with two gene copies)
• (Optional: sample with known deletion if available)

3. Reaction Setup

Use a SYBR Green-based qPCR mix with:

• 10 ng genomic DNA
• Primers (target + reference)
• Master mix
• Nuclease-free water
  Run all reactions in triplicate.

4. Thermal Cycling

Typical protocol:

• 95°C – 3 min (initial denaturation)
• 95°C – 15 sec, 60°C – 30 sec, 72°C – 1 min (40 cycles)
• Followed by melt curve analysis

5. Melt Curve Analysis

• Ensures specificity of the amplicon.
• A single sharp peak means the target was specifically amplified.
• Multiple peaks or broad curves → nonspecific amplification or primer-dimers → redesign primers.

How to Analyze Results: ΔCt, ΔΔCt, and Copy Number

Step 1: Calculate ΔCt

ΔCt = Ct(target) – Ct(reference)
This normalizes for DNA input variation.

Step 2: Calculate ΔΔCt

ΔΔCt = ΔCt(patient) – ΔCt(control)
Compares the test sample to a known normal sample.

Step 3: Calculate Copy Number

Copy number ratio = 2^(–ΔΔCt)

Example

Result: The patient has a heterozygous deletion in the target region.

Watch Out for Polymorphic Dropouts

A common cause of false positives is the presence of a SNP or indel in the primer binding site. This prevents the primer from binding efficiently, giving delayed Ct or no amplification.

It looks like a deletion, but the gene is actually present.

How to Avoid:

• Use genome browsers to check for known polymorphisms.
• Run a melt curve to catch nonspecific products.
• Use multiple primer sets if needed.

Final Thoughts

SYBR Green-based qPCR is a reliable and scalable method for confirming deletions, especially when resources are limited. It’s not a replacement for MLPA or CNV arrays, but a great validation tool when used smartly.

With careful design and interpretation, you can confidently use qPCR for deletion analysis in both clinical and research settings.

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