Services

Header

Header

Header

Custom Hybridization Probe Sets

Custom-designed DNA, RNA, PNA and affinity-enhanced hybridization probe sets for molecular diagnostics, fluorescence imaging, cytogenetics, mutation detection and spatial biology.

DNA Probes RNA Probes LNA/BNA PNA Multiplex Panels Spatial Biology

From Target Sequence to Hybridization-Ready Probe Set

Bio-Synthesis designs and manufactures custom hybridization probe sets for RNA localization, DNA detection, cytogenetics, mutation analysis, molecular diagnostics and spatial biology. Projects may range from a single fluorescent probe to large multiplex panels, PNA probes, branched DNA amplification systems or barcoded spatial transcriptomics sets.

Our scientists optimize target accessibility, probe length, hybridization Tm, probe chemistry, fluorophore selection, purification strategy and panel organization to produce hybridization probe sets tailored to your experimental objectives.

Four Key Elements of Hybridization Probe Design

Sequence Design Target accessibility, GC balance, Tm and off-target screening.
Chemistry Selection DNA, RNA, LNA/BNA, PNA, ENA/cEt and stability options.
Labeling Strategy Fluorophore, hapten, internal label or multiplex channel.
Probe Architecture Single probe, panel, bDNA, FISH or spatial barcode system.
Custom Probe Set Optimized for the target, assay and detection platform
Sequence + Chemistry + Labeling + Architecture

Complete Probe Design

Sequence review, target accessibility, probe length, Tm balance and cross-hybridization screening.

Advanced Probe Chemistry

DNA, RNA, LNA/BNA, PNA, ENA/cEt, 2′-O modifications and backbone engineering.

Fluorescent Labeling

Broad dye portfolio with terminal, internal, dual-label and multiple-fluorophore options.

Multiplex Manufacturing

Tube, plate, pooled, barcoded and project-specific probe-set formats with analytical QC.

Select Your Experimental Objective

Select the application that best matches your experiment to explore recommended probe architectures, chemistries, fluorophore strategies and purification options.

Gene Expression Probe Panels

Use multi-probe RNA panels, direct fluorescent probes or amplified architectures depending on target abundance.

DNA probes smFISH bDNA Far-red dyes
Architecture

Multi-probe panel or amplified system.

Chemistry

DNA first; LNA/BNA or PNA for difficult regions.

Purification

HPLC for labeled probes and critical panels.

Best Use

Transcript abundance and expression profiling.

RNA Localization and Imaging

Use directly labeled probe sets, RNA FISH, smFISH or PNA probes for subcellular localization.

RNA FISH smFISH PNA Alexa Fluor
Architecture

Single probe or multi-probe imaging set.

Chemistry

DNA, LNA/BNA or PNA.

Fluorophore

Bright photostable visible or far-red dye.

Best Use

Cellular localization and single-molecule imaging.

DNA Hybridization and Genomic Detection

Use DNA, LNA/BNA, PNA or longer oligo panels for genomic targets, loci and repeat regions.

DNA FISH PNA FISH Oligopaint Biotin/DIG
Architecture

Single locus probe or genomic probe panel.

Chemistry

DNA, PNA or affinity-enhanced DNA.

Label

Direct fluorophore or hapten detection.

Best Use

Chromosomal loci, repeats and genomic visualization.

Mutation and SNP Detection

Use compact high-affinity probes that maximize matched-versus-mismatched discrimination.

LNA/BNA MGB PNA ENA/cEt
Architecture

Short allele-specific probe pair.

Chemistry

MGB, LNA/BNA, PNA or ENA/cEt.

Design

Center mutation and balance probe Tm.

Best Use

SNP genotyping and rare-allele detection.

Cytogenetics and Chromosome Analysis

Use chromosome-targeted probes, telomere probes, PNA FISH or Oligopaint-style probe sets.

Telomere PNA DNA FISH Oligopaint Multiplex dyes
Architecture

Repeat probe, locus set or chromosome panel.

Chemistry

DNA, PNA or mixed affinity chemistry.

Label

Direct fluorophore or multi-color panel.

Best Use

Chromosome structure, telomeres and cytogenetics.

Spatial Transcriptomics and Highly Multiplexed Imaging

Use encoding probes, orthogonal readout probes and barcoded panels for MERFISH, seqFISH and related methods.

MERFISH seqFISH Readout probes Barcoded panels
Architecture

Encoding and readout probe system.

Chemistry

DNA with project-specific modifications.

Manufacturing

Plate-based and panel-level organization.

Best Use

High-plex spatial biology and transcript mapping.

Choose the Right Probe Chemistry

Standard DNA Probes

Flexible, scalable and compatible with a broad range of dyes and probe architectures.

Routine hybridization Multiplex panels FISH
Affinity

Moderate

Design Ease

High

Relative Cost

Lower

Best For

General probe sets and large panels.

RNA Hybridization Probes

RNA-like hybridization and strong RNA:RNA duplex formation for selected applications.

RNA targets Hybridization assays Specialty panels
Affinity

Moderate–High

Stability

Lower without protection

Design Ease

Moderate

Best For

RNA-focused hybridization systems.

LNA/BNA-Enhanced Probes

Strategic placement raises Tm and improves mismatch discrimination while allowing shorter probes.

Short targets SNP detection Structured regions
Affinity

High

Specificity

High

Typical Effect

Position-dependent Tm lift

Best For

Mutation, FFPE and difficult targets.

PNA Hybridization Probes

Neutral-backbone probes with high affinity and strong specificity for short or structured targets.

PNA FISH Telomeres Structured RNA
Affinity

Very High

Backbone

Neutral

Specificity

Very High

Best For

Short, difficult and repetitive targets.

ENA and cEt Probes

Advanced affinity chemistries for compact high-Tm probes and sequence-specific tuning.

Compact probes High Tm Advanced design
Affinity

High

Specificity

High

Design Ease

Advanced

Best For

Short or sequence-constrained probes.

PS and 2′-O-Modified Probes

Stability-oriented chemistries for difficult sample matrices and nuclease-sensitive workflows.

PS protection 2′-OMe 2′-F
Stability

High

Affinity

Variable

Use

Selective placement

Best For

FFPE and stability-sensitive assays.

Which Hybridization Technology Is Right for Me?

Recommended: RNA FISH, smFISH or PNA RNA FISH

Choose based on transcript abundance, target accessibility and spatial resolution.

Routine imaging

RNA FISH probe set

Single molecule

smFISH panel

Structured RNA

PNA or LNA-enhanced probe

Recommended: DNA FISH, PNA FISH or Oligopaint

Use probe architecture according to target size, repeat content and chromosome coverage.

Single locus

DNA or LNA-enhanced probe

Repeat/telomere

PNA FISH

Large region

Oligopaint-style panel

Recommended: LNA/BNA, MGB, PNA or ENA/cEt

Use compact probes that maximize matched-versus-mismatched discrimination.

SNP

LNA/BNA or MGB

Short target

PNA

Advanced tuning

ENA/cEt

Recommended: Branched DNA or Multi-Probe Amplification

Use amplified architectures when target abundance is too low for direct fluorescence.

Low copy

bDNA amplification

Moderate copy

Multi-probe fluorescent set

Validation

Direct probe plus amplified backup

Recommended: MERFISH, seqFISH or Barcoded Spatial Panels

Use encoding and readout systems with orthogonal barcodes and panel-level QC.

Error-robust imaging

MERFISH

Sequential imaging

seqFISH

Custom workflow

Barcoded spatial panel

Compare Hybridization Probe Technologies

Technology Specificity Sensitivity Multiplex Structured RNA Imaging Spatial Biology
DNA Probes ●●●●● ●●●●● ●●●● ●●●●● ●●●●● ●●●●●
LNA/BNA ●●●●● ●●●● ●●●●● ●●●● ●●●●● ●●●●●
PNA ●●●●● ●●●● ●●●●● ●●●●● ●●●●● ●●●●●
Branched DNA ●●●●● ●●●●● ●●●●● ●●●●● ●●●● ●●●●●
smFISH ●●●● ●●●●● ●●●●● ●●●●● ●●●●● ●●●●●
MERFISH / seqFISH ●●●● ●●●● ●●●●● ●●●●● ●●●●● ●●●●●

Representative Hybridization Probe Architectures

Single Hybridization Probe

One directly labeled probe for abundant, accessible or validation targets.

Multi-Probe Panels

Multiple probes distributed across one target to increase cumulative signal.

Multiplex Probe Panels

Color-balanced or sequence-barcoded panels for multiple targets.

PNA Hybridization Probes

Neutral-backbone probes for short, structured or difficult targets.

Branched DNA Amplification

Amplified architectures for low-copy nucleic acid detection.

RNA / DNA FISH Sets

Direct imaging probes for RNA, genomic loci and chromosome targets.

Spatial Transcriptomics Panels

Encoding and readout systems for MERFISH, seqFISH and related methods.

Custom Probe Architectures

Internal labels, multiple fluorophores, click handles, spacers and specialty formats.

Select Probe Modifications Based on Performance Goals

Affinity Enhancement

Increase Tm, shorten probes or improve mismatch discrimination.

LNA BNA PNA ENA cEt ZNA®
Use When

Targets are short, structured or mutation-sensitive.

Avoid

Excessive stabilization that reduces discrimination.

Fluorescent Labeling

Select dye family and channel according to imaging hardware, background and multiplex needs.

Alexa Fluor ATTO Cy Dyomics JF CF
Use When

Direct fluorescence or multiplex imaging is required.

Avoid

Dyes with overlapping spectra in separate channels.

Signal Generation and Amplification

Increase total signal through probe count, multiple labels, branched architectures or hapten detection.

Multiple fluorophores bDNA Biotin DIG
Use When

Target abundance is low.

Avoid

Adding poorly specific probes simply to increase signal.

Backbone Engineering

Modify charge, stability or affinity through backbone chemistry.

PS PN PACE PNA
Use When

Stability or specialized hybridization behavior is needed.

Avoid

Assuming every backbone improves every application.

Linkers and Spacers

Control steric distance between probe, fluorophore, hapten or conjugation handle.

Spacer18 HEG TEG PEG C3/C6 linkers
Use When

Labels interfere with hybridization or detection.

Avoid

Adding long spacers without a specific steric need.

Stability Enhancement

Improve resistance to nucleases or harsh sample environments.

PS 2′-OMe 2′-F Inverted dT
Use When

Samples or workflows expose probes to degradation.

Avoid

Over-modification that changes hybridization behavior.

Common Hybridization Probe Design Challenges

Weak Signal

Likely Causes
  • Low target abundance
  • Poor target accessibility
  • Insufficient probe coverage
  • Weak fluorophore
Recommended Design

Increase independent target sites, move probes away from structured regions and use a brighter dye or amplified architecture.

Chemistry

DNA first; add limited LNA/BNA or PNA when affinity is limiting.

Avoid

Adding more poorly specific probes without checking sequence quality.

High Background

Likely Causes
  • Cross-hybridization
  • Low-complexity sequences
  • Probe concentration too high
  • Over-stabilized chemistry
Recommended Design

Remove repetitive probes, reduce concentration and increase wash stringency.

Chemistry

Use standard DNA or lightly modified probes rather than heavily stabilized designs.

Avoid

Keeping probes that match related transcripts or genomic repeats.

GC-Rich Target

Likely Causes
  • High Tm
  • Secondary structure
  • Uneven probe performance
Recommended Design

Shorten probes, redistribute target sites and avoid long GC runs.

Chemistry

DNA may already be sufficient; use affinity chemistry sparingly.

Avoid

Adding LNA or cEt to an already over-stabilized GC-rich probe.

Strong Secondary Structure

Likely Causes
  • Stable stem-loop region
  • Protein-bound target
  • Probe too long
Recommended Design

Select alternative accessible regions or use several shorter probes.

Chemistry

PNA, LNA/BNA, ENA or cEt can improve binding to difficult regions.

Avoid

Over-modifying one inaccessible sequence instead of redesigning it.

SNP or Mutation Discrimination

Likely Causes
  • Probe too long
  • Mutation near terminus
  • Insufficient mismatch penalty
Recommended Design

Center the variant, shorten the probe and balance allele-specific Tm.

Chemistry

LNA/BNA, MGB, PNA or ENA/cEt.

Avoid

Over-stabilization that allows both alleles to bind.

FFPE Sample

Likely Causes
  • Fragmented target
  • Crosslinking
  • Autofluorescence
Recommended Design

Use shorter target regions, multiple independent sites and far-red dyes.

Chemistry

LNA/BNA, PNA, 2′-OMe or limited PS protection.

Avoid

Assuming chemistry alone can overcome damaged target material.

Multiplex Imbalance

Likely Causes
  • Different target abundance
  • Unequal dye brightness
  • Different probe counts
  • Spectral overlap
Recommended Design

Balance probe count, chemistry, concentration and dye assignment across the panel.

Chemistry

Keep affinity profiles comparable unless one target requires specialized chemistry.

Avoid

Using spectrally similar dyes as independent channels.

Review These Design Considerations

Target Accessibility

Avoid strongly folded or protein-bound sites.

GC Content

Avoid long GC or homopolymer runs.

Probe Length

Use the shortest sequence that meets Tm and specificity goals.

Hybridization Tm

Match probe Tm to assay conditions and panel design.

Hairpins and Dimers

Remove strong self-structure and probe-probe interactions.

Cross-Hybridization

Screen related transcripts and repetitive regions.

Variant Position

Center SNPs or mutations when possible.

Fluorophore Selection

Match dye spectra to filters and sample background.

Purification Strategy

Use high-resolution purification for labeled or critical probes.

Quality Control

Plan analytical traces, mass confirmation where compatible, OD and COA.

Need help choosing a hybridization probe strategy?

Probe Design Consultation: If you are unsure which hybridization strategy is best, our scientists can recommend the optimal probe architecture, chemistry, fluorophore selection, purification strategy and quality control plan based on your target sequence, assay format and performance objectives.

What to Send

  • Target sequence and organism
  • Application and sample type
  • Probe architecture
  • Desired chemistry or fluorophore
  • Scale, purification and QC

Quality Systems & Manufacturing Support

QMS

ISO-Supported Oligonucleotide Manufacturing

Custom hybridization probes, multiplex panels, PNA probes, FISH sets and spatial probe programs with controlled synthesis, purification, analytical QC, documentation and project-specific packaging.

ISO 9001:2015 Quality management system
ISO 13485:2016 Medical-device quality framework
ISO 14001 Environmental management system
Analytical QC HPLC/UPLC, MS where compatible, OD and COA

Selected References for Hybridization Probe Design

  1. Femino AM, Fay FS, Fogarty K, Singer RH. Visualization of single RNA transcripts in situ. Science. 1998;280:585-590.
  2. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008;5:877-879.
  3. Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X. RNA imaging with sequential FISH. Science. 2015.
  4. Moffitt JR, Hao J, Wang G, et al. High-throughput single-cell gene-expression profiling with MERFISH. Proc Natl Acad Sci USA. 2016.
  5. Eng CL, Lawson M, Zhu Q, et al. Transcriptome-scale super-resolved imaging in tissues by seqFISH+. Nature. 2019.
  6. Bio-Synthesis Technical Resources: RNA FISH Probes, PNA FISH Probes, Affinity-Enhanced Probes, MERFISH Probes, seqFISH Probes and Branched DNA Probe Systems.

Why Choose Bio-Synthesis

Trusted by biotech leaders worldwide for over 45 years of delivering high-quality, fast, and scalable synthetic biology solutions.