Multiplex polymerase chain reaction of Alu polymorphic insertions

Abstract

Alu sequences are present in humans in excess of 500,000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. Polymorphic Alu insertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)-based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to the Alu polymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five different Alu polymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimize Alu multiplex PCR.

Inference of human geographic origins using Alu insertion polymorphisms

“…While the analysis of the Alu genotype data can be accomplished relatively quickly (<20 min on a 3 GHz processor), the development of multiplex compatible systems are essential for the transition of this approach to the forensic community. Although, multiplex PCR has been successful in testing three to four Alu element insertions simultaneously [27,40], at least 25 separate PCR reactions would still be required for data collection using these manual systems. Therefore, automated multiplexed genetic systems using high throughput analysis technology are currently under development in our laboratories.…”

The Basques according to polymorphic Alu insertions

“…A dynamic type of genetic change in the human genome is the insertion by retroposition of mobile genomic elements such as the Alu family (Novick et al 1996;Rowold and Herrera 2000). Recent Alu insertions provide a novel set of DNA polymorphisms of interest for human population genetic studies (Novick et al , 1998Thomas and Herrera 1998). Some human-specific repeats (Batzer et al 1990;Batzer and Deininger 1991) and Sb2 Alu elements (Jurka 1993;Hutchinson et al 1993) have retroposed recently and have not been fixed within the human genome (Batzer et al 1995).…”

A Simple Method of Generating Complex DNA Profiles Utilizing Alu-based Markers with Applications in Forensics, Paternity, Genetic Mapping, Population Studies and Ancestry

“…A number of IF Alu variants have been identified [3,4] and therefore, combining several of these markers in a single reaction exponentially increases the possible number of genotypes without increasing the amount of reactions. A major obstacle in generating these profiles by multiplex PCR, however, is the formation of heteroduplexes, as very similar Alu elements are found in each presence variant, and limits the capability of standard Taq DNA polymerase with a variety of conditions [5][6][7]. The Failsafe system (Epicentre Biotechnologies, Madison, WI) designed for difficult or multiplex PCR has been demonstrated to work well with up to four IF Alu loci in a single reaction, referred to as an Alutetraplex, with the primary adjustment being the modification of primer concentrations [6,7].…”