As a marker for mtDNA mutations we used the “common”
deletion. This 4977-bp (mtDNA
4977) deletion, as
well as being an important cause of mitochondrial disease, has been
observed in many tissues with increasing age.
13 A modified
version of the semiquantitative PCR method of Corral-Debrinski et
al.
21 was used to estimate the proportion of the
mtDNA
4977 deletion in the total mtDNA extracted
from the retinal samples. Samples were digested in 100 μl lysis
buffer (500 mM Tris-HCl [pH 8.5]), 1 mM EDTA, 0.5% Tween 20, and 200μ
g/ml proteinase K) overnight at 37°C. After DNA extraction, the
resultant solution was phenol-chloroform purified and ethanol
precipitated using standard methodology
22 and resuspended
in 8 μl (pH 7.4) TE buffer (10 mM Tris, 1 mM EDTA) overnight at
4°C. Each sample was initially linearized using the restriction
enzyme
BamHI (1 μl enzyme and 1 μl commercially supplied
buffer) at 37°C for 90 minutes. Using serially diluted standards, we
were able to show that the method was semiquantitative under our
conditions
(Fig. 1a) .
Two dilution series were prepared for mtDNA4977 (twofold increases in dilution per step) and total mtDNA (an
initial 10-fold dilution followed by twofold increases in dilution per
step). One microliter from each dilution step was used for PCR
amplification using the following primers: for total mtDNA L3108
(nucleotide [nt] 3108-3127) and H3717 (nt3717-3701); and for
mtDNA4977, L8282 (nt8282-8305) and H13851
(nt13851-13832).
The reaction was performed in a thermal cycler (Omnigene; Hybaid, Ltd.,
Teddington, UK) under the following conditions: an initial 2-minute
denaturation step, followed by 34 cycles of denaturation (45 seconds at
94°C), annealing (30 seconds at 51°C for total mtDNA or 30 seconds
at 56°C for mtDNA4977), and extension (1 minute
at 72°C), with a final 8-minute extension at 72°C. All PCR
reactions were performed in the following mixture (50 μl): Sample DNA
1 μl, 0.6 μM forward primer, 0.6 μM reverse primer, 0.2 mM dNTPs,
5 μl 10× PCR buffer, (GeneAmp; Perkin Elmer, Norwalk CT), 0.2 μl
DNA polymerase (Amplitaq; Perkin Elmer), and 35.75 μl
sterile nanopure water. Finally, 50 μl mineral oil was added to each
tube.