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Patient DNA

Patient DNAs were obtained from the Centre for Medical Genetics, Our Lady’s Hospital
for Sick Children, Dublin, Ireland and Oxford Medical Genetics Laboratories, Oxford,
UK. DNA was extracted from peripheral blood using the Centra Puregene Blood Kit (Qiagen,
Manchester, UK) or the EZ1 Blood Kit on the EZ1 advanced XL instrument (Qiagen, Manchester,
UK) according to the manufacturers’ instructions. All samples used in this study were
previously tested for LHON mutations using PCR amplification and DNA sequencing. To
maintain patient confidentiality during this study, aliquots of residual DNA material
from the diagnostic test were labelled with the LHON mutation detected and irreversibly
anonymised. The use of patient DNA in this study has received ethical approval from
the Dublin Institute of Technology Research Ethics Committee (RN: 14–06).

Synthetic control DNA

To provide an unlimited, reliable and patient-free resource for LHON testing across
all current testing platforms as well as to allow for the development of the multiplex
PCR-restriction fragment length polymorphism (RFLP) test described in this study,
LHON control sequences were synthesised and cloned into standard plasmids by Eurofins
Genomics (London, UK) or Life Technologies (Carlsbad, USA) based on the reference
sequence NC_012920.1. A total of 6 plasmids were generated containing the 3460G, 3460A,
11778G, 11778A, 14484 T and 14484C sequences. The 3460 plasmids contained mitochondrial
sequences from 3275 to 4272, the 11778 plasmids contained mitochondrial sequences
from 11580 to 12118 and the 14484 plasmids contained mitochondrial sequences from
14449 to 15022 (Table 1). For the generation of synthetic diagnostic controls, the plasmids were combined
at a concentration of 1.5 ng/?l to generate mixes containing none or one of the primary
LHON mutations as follows; Normal control that contained no LHON mutations (a mix
of 3460G, 11778G and 14484 T plasmids at a concentration of 1.5 ng/?l), 3460A control
that contained 3460A, 11778G and 14484 T plasmids, 11778A control that contained 3460G,
11778A, 14484 T plasmids, and 14484C control that contained 3460G, 11778G, 14484C
plasmids. This information is summarised in Table 1.

Table 1. Synthetic LHON control plasmids and mixes

Primer design and PCR

Primers (Sigma Genosys, Arklow, Ireland) for the multiplex PCR-RFLP (Table 2) were designed to incorporate a MaeIII (Roche, Burgess Hill, UK, Catalogue Number
10822230001) restriction site (?GTnAC) in the presence of 3460A and 14484C mutations.
MaeIII was chosen because the 11778 mutation naturally introduces a MaeIII restriction
site and minor alterations of the PCR primers as shown in Fig. 2a and Table 2, the 3460A and 14484C mutations also introduce a MaeIII site to allow the development
of a multiplex PCR and RFLP strategy based on the modified primers and MaeIII restriction
enzyme. The sequences of the PCR products highlighting the positions of the oligonucleotice
primers and the MaeIII restriction sites are shown in the Additional files  1, 2 and 3. In Fig. 2a, alterations in the primer sequences leading to the generation of a MaeIII site are
shown in lower case. Additionally, the 3460 PCR product contains a naturally occurring
MaeIII site at position 3736 to act as an internal control of complete restriction
in the assay. This multiplex assay uses one PCR reaction and one restriction enzyme
thus allowing for a simple and cost effective assay for the 3 common LHON associated
mutations. The primers produce products as follows: 3460 F/R (333 bp), 11778 F/R (164 bp)
and 14484 F/R (236 bp), respectively. In the presence of the mutated allele, the products
change as follows: the 3460A mutation resulting in 279 bp, 28 bp and 26 bp (control
of restriction), 11778A mutation producing 135 bp, 29 bp, and 14484C mutation producing
205 bp, 31 bp, respectively. PCR was performed in a reaction containing 50 ng of genomic
DNA (or 1 ?l of the synthetic control described above) using 1 unit of Platinum Taq
polymerase (Life Technologies, Carlsbad, USA, Catalogue Number 10966–034), 100 ?M
dNTP (Life Technologies, Carlsbad, USA, Catalogue Number 10297–018), a primer mix
containing 250 ng of 3460 F/R, 60 ng of 11778 F/R and 40 ng of 14484 F/R and standard
PCR buffer containing 3.5 mM MgCl
₂
(Life Technologies, Carlsbad, USA, Catalogue Number 10966–034). PCR conditions were
95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 59 °C for 30 s and 72 °C
for 30 s followed by a final extension at 72 °C for 5 min. Restriction with MaeIII
was performed using a reaction containing 12 ?l of PCR product, 12.5 ?l of 2X MaeIII
buffer and 0.5 ?l (1 unit) of MaeIII for a minimum of 2 h at 55 °C. Restriction products
were detected by electrophoresis on a 2.5 % agarose gel (Life Technologies, Carlsbad,
USA, Catalogue Number 16500–100) containing 0.5 ?g/ml ethidium bromide (Sigma Genosys,
Arklow, Ireland, Catalogue Number E7637).

Table 2. Primer sequences for multiplex PCR-RFLP