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Design qPCR primers and TaqMan/molecular beacon probes using primer3-py. Configure probe Tm, primer-probe spacing, and hydrolysis probe constraints for real-time PCR assays. Use when designing qPCR primers and probes.
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Install and run the BD Rhapsody™ Sequence Analysis Pipeline (v3.0) on a shared cluster or remote Linux server with no root and no container runtime. Covers the self-contained install bundle, reference archives, FASTQ manifests, per-library YML generation, SLURM array execution, outputs, sample-tag demultiplexing, and the failure modes that cost hours — wrong Sample_Tags_Version on nuclei runs, uncapped Maximum_Threads, node-local scratch, and pinning a stale `latest` bundle.
Advanced single-cell multi-omics analysis including scRNA-seq, scCITE-seq, scATAC-seq, and TARGET-seq. Use when analyzing single-cell data, cell type identification, trajectory analysis, differential expression, UMAP/clustering, integrating protein and RNA modalities (TotalVI), or working with Scanpy, Seurat, scvi-tools. Includes workflows for MPN, hematologic malignancies, megakaryocyte biology.
Detects differential alternative splicing between conditions using rMATS-turbo (binomial LRT on junction counts), leafcutter (Dirichlet-multinomial GLM on intron clusters), MAJIQ V3 deltapsi/HET (Bayesian posterior on LSVs), SUPPA2 (empirical-null on TPM-derived PSI), or Shiba (junction-imbalance-corrected, 2025 SOTA at low coverage). Reports FDR-corrected significance and delta PSI effect sizes. Tools differ in statistical model, annotation dependence, calibration regime, and replicate-count requirements. Use when comparing splicing patterns between treatment groups, tissues, or disease states.
| name | primer-design-qpcr-primers |
| description | Design qPCR primers and TaqMan/molecular beacon probes using primer3-py. Configure probe Tm, primer-probe spacing, and hydrolysis probe constraints for real-time PCR assays. Use when designing qPCR primers and probes. |
| tool_type | python |
| primary_tool | primer3-py |
Reference examples tested with: BioPython 1.83+, pandas 2.2+, primer3-py 2.0+
Before using code patterns, verify installed versions match. If versions differ:
pip show <package> then help(module.function) to check signaturesIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
Design primers and internal probes for quantitative PCR using primer3-py.
"Design qPCR primers with probe" -> Generate primer pairs plus internal TaqMan/molecular beacon probes with constrained Tm and spacing.
primer3.design_primers(seq_args, global_args) with PRIMER_PICK_INTERNAL_OLIGO=1 (primer3-py)import primer3
from Bio import SeqIO
sequence = 'ATGCGTACGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG' * 3
result = primer3.design_primers(
seq_args={'SEQUENCE_TEMPLATE': sequence},
global_args={
'PRIMER_PICK_LEFT_PRIMER': 1,
'PRIMER_PICK_RIGHT_PRIMER': 1,
'PRIMER_PICK_INTERNAL_OLIGO': 1, # Design internal probe
'PRIMER_PRODUCT_SIZE_RANGE': [[70, 150]], # Short amplicons for qPCR
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 58.0,
'PRIMER_MAX_TM': 62.0,
'PRIMER_INTERNAL_OPT_TM': 70.0, # Probe Tm ~10C higher
'PRIMER_INTERNAL_MIN_TM': 68.0,
'PRIMER_INTERNAL_MAX_TM': 72.0,
'PRIMER_INTERNAL_MIN_SIZE': 18,
'PRIMER_INTERNAL_OPT_SIZE': 25,
'PRIMER_INTERNAL_MAX_SIZE': 30,
}
)
num_returned = result['PRIMER_PAIR_NUM_RETURNED']
print(f'Found {num_returned} primer/probe sets')
for i in range(num_returned):
left = result[f'PRIMER_LEFT_{i}_SEQUENCE']
right = result[f'PRIMER_RIGHT_{i}_SEQUENCE']
probe = result[f'PRIMER_INTERNAL_{i}_SEQUENCE']
probe_tm = result[f'PRIMER_INTERNAL_{i}_TM']
left_tm = result[f'PRIMER_LEFT_{i}_TM']
right_tm = result[f'PRIMER_RIGHT_{i}_TM']
product_size = result[f'PRIMER_PAIR_{i}_PRODUCT_SIZE']
print(f'Set {i}:')
print(f' Forward: {left} (Tm: {left_tm:.1f}C)')
print(f' Reverse: {right} (Tm: {right_tm:.1f}C)')
print(f' Probe: {probe} (Tm: {probe_tm:.1f}C)')
print(f' Product: {product_size}bp')
result = primer3.design_primers(
seq_args={
'SEQUENCE_TEMPLATE': sequence,
'SEQUENCE_TARGET': [100, 30], # Target region for probe
},
global_args={
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_PRODUCT_SIZE_RANGE': [[60, 100], [100, 150]], # Prefer short
'PRIMER_NUM_RETURN': 5,
# Primer parameters
'PRIMER_OPT_SIZE': 20,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 58.0,
'PRIMER_MAX_TM': 62.0,
'PRIMER_OPT_GC_PERCENT': 50.0,
'PRIMER_MIN_GC': 35.0,
'PRIMER_MAX_GC': 65.0,
# Probe parameters (TaqMan: Tm 8-10C higher than primers)
'PRIMER_INTERNAL_OPT_SIZE': 25,
'PRIMER_INTERNAL_MIN_SIZE': 18,
'PRIMER_INTERNAL_MAX_SIZE': 30,
'PRIMER_INTERNAL_OPT_TM': 70.0,
'PRIMER_INTERNAL_MIN_TM': 68.0,
'PRIMER_INTERNAL_MAX_TM': 72.0,
: ,
: ,
: ,
}
)
# Additional considerations for TaqMan probes
global_args = {
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_PRODUCT_SIZE_RANGE': [[70, 150]],
# Probe Tm should be 8-10C higher than primers
'PRIMER_OPT_TM': 60.0,
'PRIMER_INTERNAL_OPT_TM': 70.0,
# Probe should be closer to forward primer
'PRIMER_INTERNAL_MIN_SIZE': 18,
'PRIMER_INTERNAL_MAX_SIZE': 30,
# Avoid long poly-X runs in probe
'PRIMER_INTERNAL_MAX_POLY_X': 3,
}
# For SYBR Green, design primers without probe
result = primer3.design_primers(
seq_args={'SEQUENCE_TEMPLATE': sequence},
global_args={
'PRIMER_PICK_LEFT_PRIMER': 1,
'PRIMER_PICK_RIGHT_PRIMER': 1,
'PRIMER_PICK_INTERNAL_OLIGO': 0, # No probe
'PRIMER_PRODUCT_SIZE_RANGE': [[70, 200]], # Short for qPCR
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 58.0,
'PRIMER_MAX_TM': 62.0,
'PRIMER_MAX_SELF_ANY': 4, # Strict for SYBR specificity
'PRIMER_MAX_SELF_END': 2,
'PRIMER_PAIR_MAX_COMPL_ANY': 4,
'PRIMER_PAIR_MAX_COMPL_END': 2,
}
)
# For cDNA-specific amplification, target exon junction
# Mark the exon junction position
exon_junction = 150 # Position where exons meet
result = primer3.design_primers(
seq_args={
'SEQUENCE_TEMPLATE': sequence,
'SEQUENCE_OVERLAP_JUNCTION_LIST': [exon_junction], # Primer must span
},
global_args={
'PRIMER_PRODUCT_SIZE_RANGE': [[70, 150]],
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION': 4, # Min bases on each side
}
)
# Design primers for multiple targets with compatible Tms
targets = [
{'name': 'gene1', 'seq': sequence1, 'target': [100, 30]},
{'name': 'gene2', 'seq': sequence2, 'target': [150, 30]},
]
results = []
for target in targets:
result = primer3.design_primers(
seq_args={
'SEQUENCE_TEMPLATE': target['seq'],
'SEQUENCE_ID': target['name'],
'SEQUENCE_TARGET': target['target'],
},
global_args={
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_PRODUCT_SIZE_RANGE': [[70, 150]],
'PRIMER_OPT_TM': 60.0, # Same Tm for all
'PRIMER_MAX_TM': 61.0,
'PRIMER_MIN_TM': 59.0,
'PRIMER_INTERNAL_OPT_TM': 70.0,
}
)
results.append(result)
# Verify Tm with primer3's thermodynamic calculations
primer_seq = 'ATGCGATCGATCGATCGATC'
# Standard Tm
tm = primer3.calc_tm(primer_seq)
print(f'Standard Tm: {tm:.1f}C')
# Tm with specific salt conditions (match your qPCR master mix)
tm_adjusted = primer3.calc_tm(
primer_seq,
mv_conc=50.0, # Monovalent cation (K+, Na+) mM
dv_conc=3.0, # Divalent cation (Mg2+) mM
dntp_conc=0.8, # dNTP mM (reduces free Mg2+)
dna_conc=250.0, # Primer concentration nM
)
print(f'Adjusted Tm: {tm_adjusted:.1f}C')
import pandas as pd
def qpcr_results_to_df(result):
rows = []
for i in range(result['PRIMER_PAIR_NUM_RETURNED']):
row = {
'pair': i,
'forward': result[f'PRIMER_LEFT_{i}_SEQUENCE'],
'reverse': result[f'PRIMER_RIGHT_{i}_SEQUENCE'],
'fwd_tm': result[f'PRIMER_LEFT_{i}_TM'],
'rev_tm': result[f'PRIMER_RIGHT_{i}_TM'],
'product_size': result[f'PRIMER_PAIR_{i}_PRODUCT_SIZE'],
}
if f'PRIMER_INTERNAL_{i}_SEQUENCE' in result:
row['probe'] = result[f'PRIMER_INTERNAL_{i}_SEQUENCE']
row['probe_tm'] = result[f'PRIMER_INTERNAL_{i}_TM']
rows.append(row)
return pd.DataFrame(rows)
df = qpcr_results_to_df(result)
print(df)
| Parameter | Primers | TaqMan Probe |
|---|---|---|
| Length | 18-25 bp | 18-30 bp |
| Tm | 58-62C | 68-72C |
| GC% | 35-65% | 30-70% |
| Amplicon | 70-150 bp | - |
| 5' base | Any | Avoid G (quenches FAM) |