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Development of Rapid Multiplex PCR Techniques

Summary:

Forensic DNA typing is currently conducted in approximately eight to ten hours. The process includes DNA extraction, quantitation, multiplex PCR amplification, and fragment length detection. Protocols require approximately three hours for amplifying a multiplex containing 16 genetic loci. Commercial multiplex short tandem repeat (STR) typing kits are not optimized for rapid PCR thermal cycling. Techniques for the rapid PCR amplification (less than 36 min) of up to 16 regions of the human genome have now been optimized. These techniques utilize commercially available instrumentation and DNA polymerases for amplification.

Understanding PCR conditions and primer design parameters that allow for rapid multiplex amplification can be applied to research focusing on performing PCR on non-standard thermal cycling devices.

Description:

With the continuing development of miniaturization technologies such as microfluidic and micro-capillary devices there is a desire to reduce the overall time required to type DNA samples. Such miniature devices could be used for initial screening at a crime scene, at a border, or at airports. There is also growing interest in using DNA in the expanding field of biometrics. An essential component of a rapid forensic DNA test is reducing the time required for the PCR amplification of STR loci.

Surveys of fast processing polymerases working in combination with rapid cycling protocols have resulted in the development of a ‘rapid’ PCR amplification protocol. Results are obtained in less than 36 min with a standard thermal cycler employing a heating rate of 4oC/s. Capillary electrophoresis characterizations of the PCR products indicate good peak balance between loci, strong signal intensity, and minor adenylation artifacts. Genotyping results are concordant with standard amplification conditions utilizing a standard three-hour (non-rapid) thermal cycling procedure. The rapid assay conditions are robust enough to routinely amplify 0.5 ng of template DNA (~80 copies of the human genome).

Success obtained by the rapid PCR protocol for forensic DNA typing can also be applied to other PCR technologies such as nucleic acid-based diagnostics, DNA biometrics and real-time PCR.

Major Accomplishments:

Development of a protocol for rapid amplification of DNA for forensic DNA typing

Start Date:

April 1, 2007

End Date:

ongoing

Lead Organizational Unit:

mml

Source of Extramural Funding:

National Institute of Justice (NIJ) funded the work described here through an interagency agreement to the NIST Office of Law Enforcement Standards.

Customers/Contributors/Collaborators:

Dr. Daniele Podini at the George Washington University

Contact

Dr. Peter M. Vallone
peter.vallone@nist.gov
1-301-975-4872
1-301-975-8505 (fax)