Taq Mutants Engineered for Forensics

• NCJ Number: 239074
• Author(s): Milko Kermekchiev, Ph.D.
• Date Published: April 2011
• Length: 44 pages
• Grant Number(s): 2008-DN-BX-K299
• Agencies: NIJ-Sponsored
• Publication Type: Report (Study/Research)
• Award: Taq Mutants Engineered for Forensics

Annotation

This study developed and applied novel genetically engineered mutants of Taq DNA polymerase, which is highly resistant to PCR inhibitors, in directing DNA analysis of forensic samples.

Abstract

In many cases, this approach can eliminate the need to purify DNA prior to PCR and decrease the time, lower the costs, and increase the efficiency of forensic DNA testing. This goal was achieved by meeting three objectives. First, researchers developed and optimized a protocol without the DNA extraction steps for direct PCR-based typing of the human STR loci from crude samples that contain blood and soil, using the researchers' novel OmniTaq and Omni Klentaq enzymes. Second, they developed specific PCR enhancers to improve the detection sensitivity of crude samples. Third, they tested the resistance of OmniTaq and Omni Klentaq to PCR inhibitors derived from substances other than blood or soil, such as urine, semen, hair/melanin, tannins, indigo dye, bones, muscle tissue, saliva, and feces/bile salts; and they extended the application of the mutant enzymes to testing crude samples of these substances. Fourth, the researchers formulated and optimized blends of OmniTaq and Omni Klentaq with some members of the Y-family thermophilic polymerases with improved performance on damaged DNA. Preliminary tests found that the cold-sensitive hot-start mutant enzyme, CesiumTaq, performs well in STR typing, and the Omni Klentaq tends to generate more stutters; therefore preference was given to exploring CesiumTaq, along with OmniTaq enzyme. Comparative tests of direct STR typing with challenging crude samples showed that the researchers' protocols outperformed the AmpFISTR Identifiler Plus kit and to some extent the PowerPlex 16 HS kit. 24 figures, 1 table, and 37 references