Human DNA quantitation using Alu element-based polymerase chain reaction.
Human forensic casework requires sensitive quantitation of human nuclear DNA from complex sources. Widely used commercially available systems detect both nonhuman and human primate DNA, often require special equipment, and have a detection limit of approximately 0.1ng. Multicopy Alu elements include recently integrated subfamilies that are present in the human genome but are largely absent from nonhuman primates. Here, we present two Alu element-based alternative methods for the rapid identification and quantitation of human DNA, inter-Alu PCR and intra-Alu PCR. Using SYBR green-based detection, the effective minimum threshold level for human DNA quantitation was 0.01ng using inter-Alu- and 0.001ng using intra-Alu-based PCR. Background cross-amplification with nonhuman DNA templates was detected at low levels using inter-Alu-based PCR, but was negligible using intra-Alu-based PCR. These Alu-based methods have several advantages over currently available systems. First, the assays are PCR based and no additional unique equipment is required. Second, the high copy number of subfamily-specific Alu repeats in the human genome makes these assays human specific within a very sensitive linear range. The introduction of these assays to forensic laboratories will undoubtedly increase the sensitivity and specificity of human DNA detection and quantitation from complex sources.
Walker
Jerilyn A
JA
Department of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University, Baton Rouge, LA 70803, USA.
Kilroy
Gail E
GE
Xing
Jinchuan
J
Shewale
Jaiprakash
J
Sinha
Sudhir K
SK
Batzer
Mark A
MA
D020087:Alu Elements / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005554:Forensic Medicine / Q000379:methods; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D004247:DNA