Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays

Abstract

The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel-or columnbased approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics and biology. Here we demonstrate a rapid, highly sensitive method for STR analysis that utilizes DNA microarray technology. We describe two adaptations to accomplish this: the use of competitive hybridization to remove unpaired ssDNA from an array and the use of neural network classification to automate the analysis. The competitive displacement technique mimics the branch migration process that occurs during DNA recombination. Our technique will facilitate the rapid deduction of identity, length, and number of repeats for the multiple STRs in an unknown DNA sample.DNA fingerprinting ͉ DNA profiling ͉ short tandem repeat

Suspension bead array branch migration displacement assay for rapid STR analysis

“…The trained neural network evaluated test samples with a 100% success rate. Lower probabilities (0.7) of neural network output values appeared only occasionally and reflect the higher variability of measured patterns and peaks, compared with the microarray results in previous studies [10]. This however does not significantly detract from the overall good performance and ease-of-use of this method.…”

“…The principle of the branch migration displacement assay for STR analysis has been described in detail [10]. In the current study we have adapted the assay to the xMAP platform, which is outlined briefly in Fig.…”

“…This procedure enabled us to emphasize data patterns for subsequent analysis. Determination of STR lengths was performed using previously Nucleic acidsdescribed BrainMaker Professional 3.2 software that combines the use of artificial neural network (ANN) classification and math rule-based analysis [10]. In brief, the software analyzes the normalized array fluorescence raw data and determines whether the sample is homozygous (one peak) or heterozygous (two peaks) for a given STR marker, based on pattern recognition.…”

“…These innovations include the preparation of a microarray platform with probes of all possible STR lengths and the incorporation of a clamp sequence to ensure proper hybridization of the target to the probe [9]. Furthermore, sequential rounds of competitive hybridization (branch migration) are performed specifically to remove targets from those probes of unequal lengths [10] Here we describe the adaptation of the branch migration displacement assay [10] to the Luminex xMAP platform in order to overcome some of these issues. We demonstrate the utility of the technique by analyzing two STR markers in 20 genomic DNA samples with known genotype profiles.…”

“…However, such approaches have been hampered by the difficulty in differentiating between STRs that are similar in length. Previously, we described several innovations to overcome this problem [9,10]. These innovations include the preparation of a microarray platform with probes of all possible STR lengths and the incorporation of a clamp sequence to ensure proper hybridization of the target to the probe [9].…”