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Design TaqMan or SYBR Green assays.

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TaqMan vs SYBR Green

ModeProbeUse Case
taqmanYesSpecific detection, multiplexing
sybrNoCost-effective, simple

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TaqMan Assay Design

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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"

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Run

primerlab run qpcr --config qpcr_config.yaml

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Output

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

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SYBR Green Design

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Configuration

workflow: qpcr

input:
  sequence_path: "target_gene.fasta"

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

output:
  directory: "output_sybr"

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Run

primerlab run qpcr --config sybr_config.yaml

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Key Considerations

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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

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Amplicon Size

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

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Validation

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

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Next Steps