Version Compatibility

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:

• Python:

  pip show <package>

  then

  help(module.function)

  to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

qPCR Primer and Probe Design

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.

• Python:

  primer3.design_primers(seq_args, global_args)

  with

  PRIMER_PICK_INTERNAL_OLIGO=1

  (primer3-py)

Required Imports

import primer3
from Bio import SeqIO

Design Primers with TaqMan Probe

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,
    }
)

Extract Probe Results

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')

qPCR-Optimized Parameters

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,
        'PRIMER_INTERNAL_MIN_GC': 30.0,
        'PRIMER_INTERNAL_MAX_GC': 70.0,
        # Avoid G at 5' end of probe (quenches FAM)
        'PRIMER_INTERNAL_MAX_SELF_ANY': 8,
    }
)

TaqMan Probe Constraints

# 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,
}

SYBR Green Primers (No Probe)

# 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,
    }
)

Design for Exon-Spanning (Avoid Genomic DNA)

# 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
    }
)

Multiplex Primer Design

# 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)

Validate Tm Calculations

# 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')

Format qPCR Results

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)

qPCR Design Guidelines

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)

Related Skills

• primer-basics - General PCR primer design
• primer-validation - Check primers for dimers and specificity
• sequence-manipulation - Work with cDNA sequences