INNULs: A Novel Design Amplification Strategy for Retrotransposable Elements for Studying Population Variation

Abstract

Objectives: Retrotransposable elements (REs), consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), are a group of markers that can be useful for human identity testing. Until now, however, due to the inherent size difference (up to 6 kb in some instances) associated with insertion and null alleles (or INNULs), the use of REs for facilitated population studies has not been sought or practical. The size of the insertion elements (from a few hundred to several thousand bp) has proven to limit their utility as a marker because of the inefficient amplicon yield with PCR. A novel primer design now facilitates INNUL marker testing. A preliminary panel of single-locus markers was developed to evaluate the potential of typing these insertion elements. Nine INNULs (5 Alu and 4 LINEs) were typed in three major North American populations and analyzed for population genetic features. In addition, the variation of each marker among the sample populations provides insight of its potential use as individual identification or ancestral marker. Methods: INNUL markers were developed into fluorescently labeled single-loci PCR. Nine markers were developed with amplicons that were less than 180 bp in length, and, depending on the locus amplicons of the INNULs, alleles varied in size from 50 to 1 bp. This allele size is noteworthy because the insertion alleles of the 9 loci range in size from 297 to 6,195 bp. The allele distribution of the INNULs was assessed and analyzed in three major North American populations. Results: Upon observation of the distribution of the alleles in three major North American populations, the markers generally met Hardy-Weinberg expectations, and there was little evidence of detectable levels of linkage disequilibrium. Due to varying distributions of the alleles in the major population groups tested, some of the markers might be better suited for use as an individual identification marker, while others are better suited for bio-ancestral studies. Conclusions: Using the primer design strategy described in our work, SINEs and (for the first time, to our knowledge) LINEs can be utilized as markers for studying population genetic variation that is more amenable to the limitations of the PCR technique. This study lays the foundation for future work of developing a multiplex panel of INNUL markers that can be used as a single-tube assay for human identity testing utilizing small amplicons (<180 bp), which could be useful for ancient or degraded forensic DNA samples.

Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21

“…A set of three primers were designed for each marker: one forward primer and two reverse primers, one for the insertion and one for the null allele. The primer design strategies to obtain small amplicons were reported earlier [16,17]. The sex determining marker, Amelogenin, consists of one labeled forward primer and one unlabeled reverse primer.…”

“…INNULs are highly abundant in the human genome and extremely stable once inserted. These markers can be useful for human identity testing [14][15][16][17][18][19][20][21].…”

“…Degraded samples may contain fragments of DNA that are less than 165 bp in length and the quantities may be limited to sub-nanogram levels of recoverable DNA [28][29][30][31]. REs can range in size from hundreds (SINEs) to several thousand (LINEs) bp in length [14][15][16][17][18][19][20][21]. Previous attempts to use Alu sequences for identity testing capitalized on the size difference between insertion and null alleles by amplifying the entire region with the same forward and reverse primers [24].…”

“…To overcome the multiplexing difficulties due to allele size differences, the unique sequences of the Alu insertion were exploited to detect the presence or absence of Alu insertion at a given genomic position [17,18]. One unique property of these REs is a direct repeat sequence at the beginning of Alu insertion and at the end of Alu element called target site duplication (TSD) [16,17]. Figure 1 shows the primer design strategies employed to design a highly efficient multiplexed amplification system of 20 retrotransposon insertion polymorphisms and Amelogenin.…”

“…Figure 1 shows the primer design strategies employed to design a highly efficient multiplexed amplification system of 20 retrotransposon insertion polymorphisms and Amelogenin. Utilizing this strategy, one can design primers to create very small amplicons to type highly degraded DNA samples regardless of the size of Alu insertion [17,18]. Primer design strategy to type insert and native (or null) alleles with similar but not exact amplicon size differences between the two allelic states.…”