Earlier detection of breast cancer reduces mortality from this disease. As a result, the development of better screening techniques is a topic of intense interest. Contrast-enhanced dual-energy mammography (DEM) is a novel technique that has improved sensitivity for cancer detection. However, the development of contrast agents for this technique is in its infancy. We herein report gold-silver alloy nanoparticles (GSAN) that have potent DEM contrast properties and improved biocompatibility. GSAN formulations containing a range of gold:silver ratios and capped with m-PEG were synthesized and characterized using various analytical methods. DEM and computed tomography (CT) phantom imaging showed that GSAN produced robust contrast that was comparable to silver alone. Cell viability, reactive oxygen species generation and DNA damage results revealed that the formulations with 30% or higher gold content are cytocompatible to Hep G2 and J774A.1 cells. In vivo imaging was performed in mice with and without breast tumors. The results showed that GSAN produce strong DEM and CT contrast and accumulated in tumors. Furthermore, both in vivo imaging and ex vivo analysis indicated the excretion of GSAN via both urine and feces. In summary, GSAN produce strong DEM and CT contrast, and has potential for both blood pool imaging and for breast cancer screening.

The most prevalent microdeletion in humans occurs at 22q11.2, a region rich in chromosome-specific low copy repeats (LCR22s). The structure of this region has defied elucidation due to its size, regional complexity, and haplotype diversity, and is not well represented in the human genome reference. Most individuals with 22q11.2 deletion syndrome (22q11.2DS) carry a de novo hemizygous deletion of ~ 3 Mbp occurring by non-allelic homologous recombination (NAHR) mediated by LCR22s. In this study, optical mapping has been used to elucidate LCR22 structure and variation in 88 individuals in thirty 22q11.2DS families to uncover potential risk factors for germline rearrangements leading to 22q11.2DS offspring. Families were optically mapped to characterize LCR22 structures, NAHR locations, and genomic signatures associated with the deletion. Bioinformatics analyses revealed clear delineations between LCR22 structures in normal and deletion-containing haplotypes. Despite no explicit whole-haplotype predisposing configurations being identified, all NAHR events contain a segmental duplication encompassing FAM230 gene members suggesting preferred recombination sequences. Analysis of deletion breakpoints indicates that preferred recombinations occur between FAM230 and specific segmental duplication orientations within LCR22A and LCR22D, ultimately leading to NAHR. This work represents the most comprehensive analysis of 22q11.2DS NAHR events demonstrating completely contiguous LCR22 structures surrounding and within deletion breakpoints. The 22q11.2 Deletion Syndrome (22q11.2DS) is a congenital malformation disorder and the most frequent microdeletion syndrome in humans 1. It has a prevalence of about one in every 3,000 live births 1,2 and one in every 1,000 pregnancies 3. Significant medical issues present in affected individuals may include: congenital cardiac defects (~ 75%), immune deficiencies, speech/language defects, intellectual disabilities, and a 25-30% risk for developing schizophrenia in adolescence or adulthood 2. The causative deletion usually occurs as a de novo event in meiosis in one of the parents. Ninety percent of affected individuals have a hemizygous ~ 3 million base pair (Mbp) deletion in chromosome 22q11.2 2,4. The mechanism responsible for the deletion is non-allelic homologous recombination (NAHR) between surrounding chromosome 22-specific low copy repeats (LCR22s) 5,6. There are eight LCR22s, labeled alphabetically as LCRA to LCRH from centromere to telomere on 22q 6. The LCR22s are comprised of sequence modules of varying lengths containing interspersed genes and pseudogenes.

Analysis of structural variations (SVs) is important to understand mutations underlying genetic disorders and pathogenic conditions. However, characterizing SVs using short-read, high-throughput sequencing technology is difficult. Although long-read sequencing technologies are being increasingly employed in characterizing SVs, their low throughput and high costs discourage widespread adoption. Sequence motif-based optical mapping in nanochannels is useful in whole-genome mapping and SV detection, but it is not possible to precisely locate the breakpoints or estimate the copy numbers. We present here a universal multicolor mapping strategy in nanochannels combining conventional sequence-motif labeling system with Cas9-mediated target-specific labeling of any 20-base sequences (20mers) to create custom labels and detect new features. The sequence motifs are labeled with green fluorophores and the 20mers are labeled with red fluorophores. Using this strategy, it is possible to not only detect the SVs but also utilize custom labels to interrogate the features not accessible to motif-labeling, locate breakpoints, and precisely estimate copy numbers of genomic repeats. We validated our approach by quantifying the D4Z4 copy numbers, a known biomarker for facioscapulohumeral muscular dystrophy (FSHD) and estimating the telomere length, a clinical biomarker for assessing disease risk factors in aging-related diseases and malignant cancers. We also demonstrate the application of our methodology in discovering transposable long non-interspersed Elements 1 (LINE-1) insertions across the whole genome.

Optical mapping of linearized DNA molecules is a promising new technology for sequence assembly and scaffolding, large structural variant detection, and diagnostics. This is currently achieved either using nanochannel confinement or by stretching single DNA molecules on a solid surface. While the first method necessitates DNA labelling before linearization, the latter allows for modification post-linearization, thereby affording increased process flexibility. Each method is constrained by various physical and chemical limitations. One of the most common techniques for linearization of DNA uses a hydrophobic surface and a receding meniscus, termed molecular combing. Here, we report the development of a microfabricated surface that can not only comb the DNA molecules efficiently but also provides for sequence-specific enzymatic fluorescent DNA labelling. By modifying a glass surface with two contrasting functionalities, such that DNA binds selectively to one of the two regions, we can control DNA extension, which is known to be critical for sequence-recognition by an enzyme. Moreover, the surface modification provides enzymatic access to the DNA backbone, as well as minimizing non-specific fluorescent dye adsorption. These enhancements make the designed surface suitable for large-scale and high-resolution single DNA molecule studies.

In humans, the telomere consists of tandem 5′TTAGGG3′ DNA repeats on both ends of all 46 chromosomes. Telomere shortening has been linked to aging and age-related diseases. Similarly, telomere length changes have been associated with chemical exposure, molecular-level DNA damage, and tumor development. Telomere elongation has been associated to tumor development, caused due to chemical exposure and molecular-level DNA damage. The methods used to study these effects mostly rely on average telomere length as a biomarker. The mechanisms regulating subtelomere-specific and haplotype-specific telomere lengths in humans remain understudied and poorly understood, primarily because of technical limitations in obtaining these data for all chromosomes. Recent studies have shown that it is the short telomeres that are crucial in preserving chromosome stability. The identity and frequency of specific critically short telomeres potentially is a useful biomarker for studying aging, age-related diseases, and cancer. Here, we will briefly review the role of telomere length, its measurement, and our recent single-molecule telomere length measurement assay. With this assay, one can measure individual telomere lengths as well as identify their physically linked subtelomeric DNA. This assay can also positively detect telomere loss, characterize novel subtelomeric variants, haploiypes, and previously uncharacterized recombined subtelomeres. We will also discuss its applications in aging cells and cancer cells, highlighting the utility of the single molecule telomere length assay.