Detection of Y-chromosome bearing bovine sperm using laser-generated gold nanoparticle bio-conjugates

This study describes two innovative aspects of molecular biology, triplex-forming-oligonucleotides (TFOs) alone and labeled to gold nanoparticles. The thesis aims to develop a novel approach for sperm gene targeting and its applicability to sperm sex sorting, based on vital genetic sequences detection. Besides the use of other markers that recognize DNA, triplex-forming oligonucleotides represent an alternative approach for specific gene detection suitable for vital cells. The success of the triplex-based chromosome targeting is governed by the specific oligonucleotide binding. Within this thesis an abundance of triplex target sites were found on the bovine Y-chromosome, which makes the triplex hybridization a valid alternative for Y-chromosome bearing sperm detection. Three candidate TFOs, with a length of circa 20 bases, were analyzed for hybridization efficiency on the bovine Y-chromosome. Better triplex hybridization efficiency was achieved using more probes simultaneously. Not all Y-chromosome sperm nuclei were detected by TFOs, most likely due to the intrinsic condensed DNA structure. This study confirms that TFOs with a low content of cytosine have a higher hybridization temperature. To define TFOs hybridization for future studies, it is necessary to consider further variables, such as time of incubation of target DNA and TFO probes, temperature, TFO’s sequence and 5- methylcytosine content, buffer and bovine individual sequence variations. Gold nanoparticle conjugated with TFOs can hybridize to genomic DNA free in solution and lead to a surface plasmon resonance shift. The use of the conjugate in sperm chromatin resulted in a non-significant signaling. More studies are needed to identify different sized AuNPs (from 2 nm to 50 nm) conjugates and their potential use in a condensed chromatin structure.