Estimating the integrity of aged DNA samples by CE

Abstract

A CE/UV method was developed to separate by a micellar system the four DNA bases and other five purinic-pyrimidinic compounds (5-methyl-cytosine, uracil, xanthyne, hypoxanthyne and 5-bromo-uracil). Selectivity, precision, accuracy and sensitivity were assessed and proved to be suitable for the analysis of the primary structure of DNA. This method was adopted to study 16 aged samples including two Egyptian mummies, formaldehyde-fixed paraffin-embedded tissues and other forensic specimens. Lower relative values of the four canonical unmodified DNA bases (uDNAb) and more complex pherograms were found in the aged samples when compared with the modern controls. The results of the CE analysis, together with those obtained by classical molecular methods (agarose gel electrophoresis, DNase I and RNase A assays, and UV spectrophotometry), were finally evaluated for assessing the reliability of STR typing. Since samples with low uDNAb showed no amplification or unreliable STR profiles, the uDNAb value is discussed as a further quality criterion in the evaluation of the genetic data obtained from aged samples.

Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography

“…The compounds investigated are small molecules having properties relevant for biomedical 2, 4–7, 11–13 and pharmaceutical studies 8–10, 14 (Table 1). In particular, five bases and the corresponding nucleosides are constituents of DNA and RNA.…”

“…In particular, five bases and the corresponding nucleosides are constituents of DNA and RNA. Hypoxanthine was added because it is obtained from adenine by a metabolic route, which involves oxidative deamination (like uracil which can originate from oxidative deamination of cytosine 6), and inosine because it is a hypoxanthine‐corresponding nucleoside.…”

“…In the last 30 years, there has been increasing request of methods for the separation of nucleic acid constituents, nucleotides, nucleosides, and bases, as well as their degradation products, metabolites, and modified synthetic analogs 1. The main research areas concerned have been genomics 2, pharmacology and toxicology 3, clinical chemistry 4, forensic 5, 6 and evolutionary genetics 7, and pharmaceutical chemistry 8–10. A number of pyrimidine and purine analogs, originating from mutations have been studied in biological specimens as disease biomarkers 11, and for evidencing changes of the structure of nucleic acids 12, 13.…”

Type-Specific Human Papillomavirus Distribution in Invasive Squamous Cervical Carcinomas in Tunisia and Vaccine Impact

“…DNA fragmentation in formalin fixed paraffin embedded tissues are significantly more frequent compared to fresh tissues so DNA integrity usually decreases overtime in those samples and the probability to get false negatives increases ( Mc Sherry et al, 2007;Fattorini et al, 2009;Webersinke et al, 2013). This can be overcome in part by the use of the general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequence (De Roda et al, 1995;Lee, 2012) or the use of a nested PCR performed with MY09/MY11 and GP5+/GP6+ biotinylated primers as we did in our study (Carvalho et al, 2010).…”

Capillary electrophoresis analysis of DNA primary structure: Toward a quality control test for the reliability of the STR-typing from forensic specimens

“…These undesirable PCR results may represent important sources of mistyping potentially giving false STR profiles, as it is impossible to distinguish them from authentic replicates of the original DNA. Capillary electrophoresis (CE) has been shown to be a powerful tool to describe the DNA primary structure and to study DNA damage, identifying both canonical and un-canonical DNA bases [2]. The aim of the present study is to evaluate if the CE analysis, together with other tests routinely applied in forensic DNA investigations, can be concurrently studied in order to bring to trustful decisions on the reliability of STR profiles obtained from degraded DNA samples.…”