Hyb & Seq™ is a specific application of the Design-On-Demand Service. The product increases efficiency and accuracy over Tm-based hybridization for the following applications:

• Detection of variable genotypes
• Detection of SNPs
• Re-sequencing genes on chip
• Hybridization of highly variable or difficult gene segments

The process is unique in its use of an on-chip enzymatic method of DNA synthesis and ligation where the discrimination is achieved at the enzymatic step. This ligation reaction occurs only for perfect matches between the probes on the chip and the synthesized target DNA with the incorporated tag and label. Sequences with mismatches do not get ligated and the label gets washed away. Therefore, signal is only generated for perfect matches. CombiMatrix offers the opportunity to custom design the probes on our CustomArray™ chips with no minimum order or set-up costs. The turn-around time for this custom service is typically less than 30 calendar days.

The customer needs to prepare an amplified target DNA fragment of interest with the incorporated tag sequence at the end. The resultant target DNA should be single-stranded.

1. The customer will send CombiMatrix the sequence of interest indicating which positions need to be analyzed.
2. CombiMatrix will then create the CustomArray™ containing 4 probes for each indicated sequence position. The 5’ termini of these 4 probes will vary (A, T, G, or C).
3. CombiMatrix will send the CustomArray™ to the customer.
4. The customer will hybridize the Custom Array™ with the prepared target DNA in the presence of a labeled oligonucleotide (corresponding to the tag incorporated into the target DNA) and two enzymes, DNA polymerase and ligase.
5. The probes with the perfect match of 5’ termini to the target DNA will ligate to the extended DNA containing labeled tag oligonucleotide and produce high intensity signals. The non-matching probes will remain dark as the labeled tag oligonucleotide will be washed away.
   

Click here to view an illustration of the process.

• Fast Assay (1-2 hours)
• Rapid re-sequencing of DNA
• Quick verification of results
• Accurate
• High conversion rate >95%
• Excellent call rate
• Results are more easily interpreted than with standard Tm-based hybridization
• Flexible
• Overcomes problems associated with standard hybridization
  • Variable optimal hybridization conditions due to variable Tm and oligo length
  • Mismatch detection in a GC-rich environment
  • Detection of difficult mismatches such as G/G, G/T
• Standard sequencing on chip without costly auto sequencer
• No need to directly label DNA target
• Labeling is accomplished using common tag primer that is extended by polymerase reaction on the CustomArray™
• CombiMatrix CustomArray™ chips ship with hybridization caps and clamps
• CustomArray™chips can be read on most standard laboratory scanners

Specifications

Hyb & Seq™ Protocol
in .pdf format

Hyb & Seq™ Workflow
in .pdf format

Hyb & Seq™ FAQ

Supporting Graphs
in .pdf format

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