Fluorescence Analysis of Expanded Trinucleotide Repeat Heterogeneity after in vitro PCR Amplification

The expansion of trinucleotide repeats in human genomic DNA manifests into multiple neurodegenerative diseases (Amrane et al., 2005). At least nine human diseases stem from the expression of expanded trinucleotide repeats (Box, 2007). Simple sequences such as, 5’CAG/CTG’3 repeats, contain a potential pathogenicity once expanded past their original lengths; which is exemplified in Huntington’s Diseases and Myotonic Dystrophy. Once expanded CAG repeats are transcribed into RNA, these transcripts are translated, and the mal functioning proteins can lead to severe cell damage. Furthermore, the repeats can be passed down through generations, and in each germline, continuously expanding, gaining more repeated units (Orr & Zoghbi, 2007)-(Hartenstine, Goodman, & Petruska, 2000). Moreover, an increasing length of CAG repeats is directly correlated with an increase in the severity of the aforementioned diseases (Orr & Zoghbi, 2007)-(Hartenstine et al., 2000). In addition, the mechanisms by which elongated CAG repeats operate in vivo remains unknown; however, past literature denotes structural and functional aspects of CAG tracts (Zhang, Huang, Gu, & Li, 2012)-(Broda, Kierzek, Gdaniec, Kulinski, & Kierzek, 2005). These past studies were performed on shorter repeat lengths of 10-80, while in vivo, CAG sequences reach over 100 repeats in length (Petruska, Arnheim, & Goodman, 1996)-(Hartenstine, Goodman, & Petruska, 2002). The heterogeneity of these repeats reflect the innate replication difficulties present in in vitro studies and physiological conditions in Huntingtins and Myotonic Dystrophy (Lin & Wilson, 2011)- (Cooper, 2009). Petruska hypothesizes that when CAG repeats are being replicated, the strand forms structures that facilitated Taq’s ability to jump over sequences and leave shortened replicons (Petruska et al., 1996). Furthermore, we aim to detect the heterogeneity of (CAG)n products after PCR amplification. By initially amplifying repeats 10-150 with long primers, we are able to verify the change in CAG sequence length after cutting the primers off with an enzymatic double digestion. In addition, (CAG)10-150 were amplified once more with smaller series of primers: this enabled us to view how increasing CAG length induces a more heterogeneous product after PCR.CAG 10 through 60 show strong bands on agarose gels; however, the bands become less intense, and eventually degrade into complete smears with increasing length. This is seen in repeats 100, to 120, and then 150. The small primers allow us to view this degradation. The smear of (CAG)150 displays a variety of repeats at different lengths; and a way to detect this variety is through a binding assay. SYBR Green is a chemical agent that fluoresces once bound to double stranded DNA. Doxorubicin- also called Daunomycinone- a well-known anticancer drug, naturally fluoresces. Once bound to double stranded DNA, Doxorubicin’s fluoresces is quenched (Das & Kumar, 2013). Utilizing these two fluorophores will allow us to detect heterogeneity after PCR amplifications (Ning et al., 2015)-(Chan et al., 2012). By titrating in select CAG repeats after PCR we can record changes in fluorescence spectra and determine whether the amplicons are the correct length (Ning et al., 2015)-(Gatchel & Zoghbi, 2005). the Fluromax-3 Fluorimeter, we can record the fluorescence quenching of Doxorubicin by titrating in select CAG sequences (Ning et al., 2015)-(Gatchel & Zoghbi, 2005)