A preliminary study on the incidence of heteroplasmy in mitochondrial DNA from vitreous humour.
Vitreous humour is routinely sampled in Forensic Medicine as several post-mortem analyses can be performed. However, it is not used for DNA analyses probably due to its scarce cellularity. In these samples, in which the study of nuclear DNA is difficult, the analysis of mtDNA is an alternative approach. The aim of this study was to investigate the utility of vitreous humour for forensic identification purposes. Samples were collected during vitrectomy from retinopathy patients, in collection bags with saline solution. Blood samples were also obtained in order to contrast results. Before DNA organic extraction, several centrifugation steps were needed to concentrate the vitreous humour samples. Unlike blood, direct amplification of 400-bp fragments of the hipervariable regions I and II (HVI and HVII) was not successful, possibly due to damage to the DNA strand caused by the surgery conditions (UV radiation, oxidative stress). Therefore, amplification of two overlapping fragments for each control region was performed in vitreous humour. In order to eliminate undesired products, all samples were purified by an enzymatic method. Thereafter, mtDNA fragments were sequenced using dye terminators in a MegaBACE 500 capillary sequencer. Sequences of HVI and HVII of approximately 400 bp were obtained from all samples. The sequences obtained from each patient matched almost perfectly those from blood. In summary, herein we describe for the first time a methodology suitable for the mtDNA analysis of vitreous humour samples.
Sosa
C
C
Department of Forensic Medicine, Faculty of Medicine, University of Zaragoza, C/Domingo Miral s/n, Zaragoza 50.009, Spain. cecis@unizar.es
Abecia
E
E
Casalod
Y
Y
Baeta
M
M
Núñez
C
C
Luna
A
A
Pérez-Cárceles
M D
MD
Martínez-Jarreta
B
B
D000328:Adult; D022801:Complementarity Determining Regions / Q000235:genetics*; D016172:DNA Fingerprinting; D053938:DNA Fragmentation; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D014822:Vitreous Body / Q000378:metabolism* / Q000473:pathology
D022801:Complementarity Determining Regions; D004272:DNA, Mitochondrial