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

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.

A novel set of DIP-STR markers for improved analysis of challenging DNA mixtures

“…To evaluate the discrimination power of DIP-STRs, the theoretical estimate of the DIP-STR informativeness (I) previously published [6] was used. This formula allows calculating the occurrence of informative genotypes based on the presence of DIP alleles unique to the minor DNA contributor, I = 2s 2 l 2 + 2s 3 l + 2sl 3 .…”

“…1). We originally described an initial set of DIP-STRs to empirically validate the high sensitivity of the method in resolving unbalanced mixed samples [6][7][8]. This study showed that because of the allele-specific amplification, these markers are about 100 times more sensitive in detecting DNA mixtures than autosomal STRs currently used for DNA profiling.…”

“…To reduce the number of cases where the DNA of interest is actually collected, but remains undetected, we proposed a novel compound genetic marker composed of a DIP tightly linked to an STR polymorphism, named DIP-STR [6]. These markers are capable of targeting a minor DNA in the presence of a 1000-fold excess of the major DNA based on a unique design of the PCR primers.…”

Sequence-based definition of eight short tandem repeat loci located within the HLA-region in an Austrian population

“…The analysis of the sequence polymorphisms in repeat and flanking regions found at some loci has the potential to increase their discriminative power if appropriate techniques (e.g. DIP-STR typing, massively parallel sequencing) are applied [36,37]: the D6S2906 alleles *10-*16 consisted of two or three subgroups as defined by SNPs in their flanking regions. The more frequent alleles *10 and *12 (0.189 and 0.198, respectively) could be split up into allelic subgroups found in a 1:3:2 and 1:3 relation, respectively according to sequencing results.…”

Chimerism in Humans after Bone Marrow Transplantation: A New Challenge for Forensic DNA Experts

“…When DNA profiling technique is being performed using any biological specimen from the individual, it promises the correct and unambiguous individualization, but in certain cases the forensic scientist may encounter the problem of mixed or completely mismatched DNA profile of a single individual [1,2]. STR's (short tandem repeat) may lead to erroneous conclusion when the source of the biological material being analyzed is from a person who is a genetic chimera.…”