WARF: P090366US02

Double-Strand DNA Break Repair in Vitro – Forensic and Genomic Applications


Michael Cox, John Battista, Audrey Klingele, Joseph Piechura

The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing a method for repairing double-strand DNA breaks in vitro to enable the sequencing or typing of DNA that may be poor quality, of limited availability or in a complex mixture of contaminating DNA sequences.
OVERVIEWSince the 1980s, DNA typing of biological evidence has played a central role in forensic science.  This technology has helped solve crimes nationwide, and also has helped exonerate wrongly accused individuals. 

DNA genotyping is based on PCR amplification and electrophoretic analysis of short tandem repeats (STRs).  Current forensic DNA technology requires 0.2 to 2 nanograms of duplex DNA that is at least 100 to 150 bp long.  However, many samples fail to meet these requirements because the DNA has been exposed to damaging environmental conditions.  For example, a double-strand break between an STR and the site targeted by a PCR primer prevents the generation of a signal in forensic DNA typing. Because conventional techniques for analyzing STRs do not work for this damaged DNA, a need exists for new DNA sequencing techniques that are not hampered by DNA quality.
THE INVENTIONUW–Madison researchers have developed a method for the in vitro joining of two DNA fragments that have homologous DNA sequences through a simplified process of DNA double-strand break repair.  This approach enables the sequencing of poor quality or minimally available DNA that may be in a complex mixture of contaminating DNA sequences.  It requires only three proteins or their homologues: RecA protein, single-stranded DNA binding protein (SSB) and DNA polymerase I.  The proteins preferably are obtained from E. coli

In this method, a single-stranded DNA probe with some homology to the target DNA is combined with RecA protein and SSB.  Then the target duplex DNA molecule, which has a double-strand break and is not super-coiled, is added to the mixture.  The single-strand probe invades the doubled-stranded target DNA.  When DNA polymerase is added with dNTPs, it extends both strands to create a double-stranded DNA molecule in which the two fragments have joined.  PCR reactions then can be carried out using primers that bind the probe and target DNA.  This will allow amplification of STRs, if there are lesions in the DNA close to an STR.
  • Forensic analysis of DNA
  • Sequencing ancient DNA
  • Filling in gaps in genome sequencing projects
  • Obtaining sequence information from unculturable microbes in metagenome analysis
  • Enables sequencing of poor quality DNA
  • Capable of separating sequences of interest from everything else and manipulating them in isolation
  • RecA significantly increases the speed at which the probe DNA identifies its target.
  • The use of homologous DNA sequences means that DNA associated with contaminating biological material will not be incorporated into the new construct.
  • The use of a capture ligand facilitates purification and automation.
  • For forensic applications, this method targets only DNA sequences adjacent to the STR and not the STR itself to generate accurate DNA typing signals in cases where none were possible prior to repair. 
  • Does not involve the use of enzymes that could alter properties of STRs that are critical to analysis
  • Prevents additional degradation or damage to the DNA sample of interest
  • Does not require super-coiled DNA, making it useful for forensic or ancient DNA samples, which are almost never super-coiled
  • Does not require the proteins gyrase or DNA PolIII
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Contact Information
For current licensing status, please contact Jennifer Gottwald at or 608-960-9854.
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