About BRCA2
The BRCA2 gene (BRCA2 DNA Repair Associated) on chromosome 13q13 Germline loss-of-function mutations in BRCA2 confer lifetime breast cancer risk of ~69% and ovarian cancer risk of ~17%. Key targets include Exons 2–27 for comprehensive mutation screening.
Mutations: Truncating, frameshift, 6174delT, 999del5 | GC content: 41% | Target exons: Exons 2–27
BRCA2 Primer Design Challenges
- Very low GC content (41%): BRCA2 has among the lowest GC content of cancer genes, causing weak primer-template stability
- Large coding sequence: 27 exons spanning 11.4 kb require 120+ tiled amplicons for full mutation screening
- Multitude of founder mutations: Population-specific frameshifts (6174delT in Ashkenazi, 999del5 in Icelandic) need targeted genotyping
Recommended Primer Design Parameters for BRCA2
| Parameter | Standard Exons | Low-GC / AT-Rich Regions |
|---|---|---|
| Primer length | 20–22 nt | 22–25 nt |
| GC content | 45–55% | 50–60% |
| Tm | 58–62°C | 60–65°C |
| Amplicon size | 150–300 bp | 180–350 bp |
| Annealing temp | 58–60°C | 60–64°C (touchdown) |
| PCR additive | Standard | Longer primers (24–28 nt), lower annealing Tₘ |
Key SNPs to Avoid in Primer Binding Sites
When designing BRCA2 primers, avoid these clinically significant variants:
- rs80359550 (6174delT) — Ashkenazi Jewish founder frameshift in exon 11
- rs80359671 (999del5) — Icelandic founder mutation in exon 9
Clinical Validation Required
All BRCA2 primers designed with VigyanLLM are for research use only. Clinical diagnostic applications require additional wet-lab validation, Sanger sequencing confirmation, and regulatory approval before patient use.
Recommended Primer Sequences for BRCA2 — Engine-Designed on NM_000059
The three pairs below were produced by VigyanLLM's automatic primer design engine directly against the Homo sapiens BRCA2 RefSeq mRNA NM_000059 (11954 nt), under the engine's default constraints: 18–25 nt primers, Tm 57–72 °C (optimum 62 °C), GC 40–60%, and an 80–1500 bp amplicon (optimum 150 bp). Every oligo was then re-checked back against the accession — each forward primer matches the sense strand at its stated 1-based position and each reverse primer matches the reverse complement ending at the amplicon's final base (3 of 3 exact matches for this page).
| Pair | Forward (5′→3′) | Reverse (5′→3′) | Tm fwd / rev | GC fwd / rev | Amplicon |
|---|---|---|---|---|---|
| 1 | 5′-TCAGTGGTATGTGGGAGTTTG-3′ | 5′-TGGGTGGTGTAGCTAAAGAAC-3′ | 58.2 / 58.2 °C | 47.6 / 47.6% | 136 bp (nt 671–806) |
| 2 | 5′-ACCTCAGCGTTTGTGTATCG-3′ | 5′-AGTGTTGGGATTACAGGCATG-3′ | 58.9 / 58.9 °C | 50.0 / 47.6% | 160 bp (nt 10583–10742) |
| 3 | 5′-TGGAGATGGAGTCTTGCTTTG-3′ | 5′-AGCTACTAGGGAGGCTTGAG-3′ | 58.6 / 58.6 °C | 47.6 / 55.0% | 118 bp (nt 11487–11604) |
Pair 1 leads with a forward/reverse Tm spread of 0.0 °C, no stable hairpin at 37 °C (ΔG ≥ 0.0 kcal·mol−1 on either oligo), self-dimer Tm at most -11.3 °C, no cross-dimer signal, no pair warnings, and a Primer3 pair-quality score of 100.0/100; the suggested starting annealing temperature is 53.2 °C. Pairs 2 and 3 are alternates from the same run — compare their Tm, GC and product columns above against your chemistry (dye-based or probe), then confirm every candidate in silico and on a gel before use.
Specificity: not confirmed.
The engine also launched an automated Primer-BLAST pass (Homo sapiens, RefSeq mRNA), but its report parser returned no expected-amplicon hit — an inconclusive result, not a confirmation. Before ordering, run NCBI Primer-BLAST on each final pair yourself. For genomic-DNA templates this matters even more: an mRNA-based check cannot see processed pseudogenes, repeats or pseudogene-derived off-targets.
How to read this table: these are default whole-transcript designs — they demonstrate what the engine produces under standard constraints and are not positioned over the clinically relevant variants discussed below; to land an amplicon on a specific codon or repeat, design against that region deliberately. Thermodynamics come from Primer3's nearest-neighbor engine (SantaLucia 1998). Example output only: validate each candidate experimentally (gel sizing, efficiency series, melt-curve or probe check) before relying on it.
Design Guidance for BRCA2 Assays
BRCA2 tethers and loads RAD51 during homologous repair of double-strand breaks; germline variants raise breast, ovarian, prostate and pancreatic cancer risks. The best-known founder variant is 6174delT (c.5946delT), a frameshift in exon 11 enriched in Ashkenazi Jewish populations, and the Icelandic 999del5 founder variant is another common screening target.
The coding sequence is unusually long — 3,418 amino acids encoded across 27 exons — and the genomic locus is far larger than the 11,954-nt transcript because of its large introns. Practical consequences: gDNA amplicons must fit within individual exons, while cDNA assays can span exon junctions (useful both for isoform resolution and for excluding genomic carry-over). As with BRCA1, exon-level copy-number changes are invisible to standard PCR and need an independent method such as MLPA. For founder-variant screening, put the altered base inside a short amplicon — short matters for the degraded DNA of archival samples — and re-confirm specificity against your actual template before ordering.
Before you order: re-check both binding sites against dbSNP and gnomAD — a common variant under one primer causes allele dropout and mysterious assay failures — confirm specificity in silico for the template you will actually use, run an annealing gradient around 53.2 °C (the engine's starting suggestion for pair 1), verify a single band of the expected size on a gel, and record primer sequence, lot and efficiency with the run as MIQE requires.
References
- NCBI Nucleotide — RefSeq NM_000059 (Homo sapiens BRCA2 mRNA).
- NCBI Gene — BRCA2 (Homo sapiens).
- SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460–1465.
- Untergasser A, Cutcutache I, Koressaar T, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res 2012;40:W115–W118.
- Ye J, Coulouris G, Zaretskaya I, et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. Nucleic Acids Res 2009.
- Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611–622.
Design BRCA2 Primers Now
Pre-configured with BRCA2-specific parameters. Use our primer design, Tm calculator, and GC content tools for optimal results.