qPCR Design Tutorial

Tutorial: qPCR Design Tutorial

Design TaqMan or SYBR Green assays.

TaqMan vs SYBR Green

Mode	Probe	Use Case
taqman	Yes	Specific detection, multiplexing
sybr	No	Cost-effective, simple

---

TaqMan Assay Design

Configuration

workflow: qpcr

input:
  sequence_path: "target_gene.fasta"

parameters:
  mode: taqman  # Default
  
  # Primer parameters
  primer_size: {min: 18, opt: 20, max: 24}
  tm: {min: 58.0, opt: 60.0, max: 62.0}
  
  # qPCR requires short amplicons
  product_size_range: [[70, 150]]
  
  # Probe parameters
  probe:
    size: {min: 18, opt: 24, max: 30}
    tm: {min: 68, opt: 70, max: 72}

qc:
  mode: strict  # Recommended for qPCR

output:
  directory: "output_qpcr"

Run

primerlab run qpcr --config qpcr_config.yaml

Output

{
  "forward": {
    "sequence": "ATGAGTAAAGGAGAAGAACT",
    "tm": 58.5
  },
  "reverse": {
    "sequence": "GCCGTGATGTATACATTGTG",
    "tm": 59.2
  },
  "probe": {
    "sequence": "TGTTGAATTAGATGGTGATGTTAA",
    "tm": 70.1
  },
  "amplicon_size": 95
}

---

SYBR Green Design

Configuration

workflow: qpcr

input:
  sequence_path: "target_gene.fasta"

parameters:
  mode: sybr  # No probe
  product_size_range: [[80, 200]]

output:
  directory: "output_sybr"

Run

primerlab run qpcr --config sybr_config.yaml

---

Key Considerations

Probe Design Rules

• Tm 8-10°C higher than primers
• No G at 5’ end (quenches fluorescence)
• Avoid long G runs (>4)
• 18-30 bp length

Amplicon Size

• 70-150 bp ideal for qPCR
• Short = higher efficiency
• Long = better sensitivity

---

Validation

primerlab insilico \
  -f "ATGAGTAAAGGAGAAGAACT" \
  -r "GCCGTGATGTATACATTGTG" \
  -t target_gene.fasta

---

Next Steps

• Off-target Analysis
• PCR Walkthrough
• Troubleshooting