Background

Testing for a single-copy or low-copy sequence (target) in DNA or RNA is commonly done by measuring the degree of hybridization between that known target and a labeled molecular probe complementary to some portion of the target.

By itself, that approach can produce distorted results because probes often contain extraneous repetitive sequences that bind to complementary repetitive sequences flanking the target and artificially elevating the hybridization signal.

One counterstrategy is to block the flanking regions by pre-hybridizng them with a reagent enriched in common repetitive sequences. C₀t-1 DNA is a often used as the blocking reagent.

But C₀t-1 DNA is a complex mixture containing extraneous low-copy sequences, and one study found how their binding to probes can lead to a different type of artificially elevated hybridization signal.

The Innovation

Researchers at Children’s Mercy Hospital have devised a class of blocking reagents that mitigate both forms of hybridization signal distortion.

In this innovation, repetitive sequences flanking the target are identified and complementary sequences are synthesized. In the pre-hybridization step, this synthetic repetitive DNA (srDNA) acts as a blocking reagent, like C₀t-1 DNA. But unlike C₀t-1 DNA, srDNA contains few (or no) extraneous low-copy sequences to bind to the probe in the hybridization step.

The result is a probe hybridization test that more accurately signals the presence or absence of the target DNA or RNA sequence.

Applications

• Alternative to C₀t-1 DNA in probe hybridization tests that require blocking of extraneous repetitive sequences
• Alternative to C₀t-1 DNA when blocking is desired but signal distortion from extraneous low-copy sequences in the probe are a concern
• Genomics research, DNA or RNA
• Genetic testing and diagnosis, DNA or RNA

Advantages

• Like C₀t-1 DNA, srDNA suppresses signal distortion from repetitive sequences flanking a target gene
• Unlike C₀t-1 DNA, srDNA has no extraneous low-copy sequences to distort the hybridization signal
• Can be optimized to block repetitive sequences typical in a specific organism

Status

• Publication: H. Newkirk, J. Knoll, and P. Rogan, Nucleic Acids Research, 2005, Vol. 33 No. 22 e191
• U.S. patent #7,833,713; foreign applications pending
• Seeking partner(s) to license and commercialize

File Number: 2006-007