DIP – STR : Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures

Vincent Castella,Joëlle Gervaix,Diana Hall

doi:10.1002/humu.22280

Vincent Castella, Joëlle Gervaix + Show 1 more

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https://doi.org/10.1002/humu.22280

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Abstract

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP–STR that relies on pairing deletion–insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP–STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP–STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.

Highlights

A theoretical estimate of the deletion–insertion polymorphisms (DIP)–short tandem repeats (STR) informativeness is possible given the fact that the occurrence of informative genotypes depends on the presence of DIP alleles, which are unique to the minor DNA contributor
To circumvent the problem of the major DNA contributor masking the minor DNA of a mixture, we propose here a PCR-based method characterized by allele-specific primers capable of targeting DNA sequences, which are unique to the minor DNA
Because biallelic markers have reduced information content, we propose the selection of DIP linked to STR to form a compound marker termed DIP–STR

Summary

Introduction

Genetic polymorphisms such as short tandem repeats (STR) and single-nucleotide polymorphism (SNP) are commonly used in forensic identity testing [Gill et al, 1994, 2001; Jobling and Gill, 2004; Kayser et al, 1997; Moretti et al, 2001; Tully et al, 2001] and medical genetics [Goate et al, 1991; Hastbacka et al, 1992; Houwen et al, 1994; Wooster et al, 1995]. Characterizing unbalanced DNA mixtures has been associated with the analysis of biological stain for forensic identification purposes. Challenging mixed stains may be derived from samples including, but not limited to, clothing, hair, skin, or items the perpetrator may have touched. Because these samples are likely to carry small quantities of the perpetrator’s DNA mixed with a large amount of the victim’s DNA, the limit of resolution of currently used markers may have dramatic consequences for justice. Several fields of medical genetics recently expressed a paramount need for tools that enable the analysis of unbalanced DNA mixture occurring in vivo, referred as DNA microchimerism (less than 1% of foreign cells). Few examples are the DNA microchimerism associated to pregnancy, which is caused by the transient circulation of minute quantities of fetal DNA in the maternal blood [Lo et al, 1998; Tjoa et al, 2006] or the trace quantities of donor’s DNA in the body fluids (blood and urine) of transplanted patients [Chok et al, 2002; Gadi et al, 2006; Moreira et al, 2009; Pujal and Gallardo, 2008]

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