Nested PCR Design Tutorial

Tutorial: Nested PCR Design Tutorial

Nested PCR is a two-step amplification technique that increases specificity. PrimerLab supports both Nested PCR and Semi-Nested PCR design.

Concept

Outer PCR:
5'────[OUTER_FWD]═══════════════════════════[OUTER_REV]────3'

Inner PCR (Nested):
          5'────[INNER_FWD]═══════════[INNER_REV]────3'

Semi-Nested:
          5'────[INNER_FWD]═══════════════════[OUTER_REV]────3'

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CLI Usage

Nested PCR Design

primerlab nested-design --sequence input.fasta --format json

Options:

Option	Description
--sequence	Input FASTA file
--outer-size	Outer product size (default: 400-800 bp)
--inner-size	Inner product size (default: 150-300 bp)
--format	Output format (json/text/csv)

Semi-Nested PCR Design

primerlab seminested-design --sequence input.fasta --format json

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Python API

from primerlab.core.variants import NestedPCRDesigner

designer = NestedPCRDesigner()
result = designer.design(
    sequence="ATCGATCG...",
    outer_product_range=(400, 800),
    inner_product_range=(150, 300)
)

print(result.outer_primers)
print(result.inner_primers)

---

Example Output

{
  "outer_primers": {
    "forward": "ATCGATCGATCGATCGATCG",
    "reverse": "GCTAGCTAGCTAGCTAGCTA"
  },
  "inner_primers": {
    "forward": "TGCATGCATGCATGCATGCA",
    "reverse": "CGATCGATCGATCGATCGAT"
  },
  "outer_product_size": 650,
  "inner_product_size": 220
}

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Best Practices

1. Outer primers should flank the region of interest
2. Inner primers should bind within the outer amplicon
3. Maintain 3-5°C Tm difference between outer and inner
4. Avoid overlapping primer binding sites

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See Also

• PCR Workflow
• CLI Reference: nested-design