Biomedical Engineering Reference
In-Depth Information
Table 2
Sequences of Allele-Specific Oligonucleotide Probes
Probe sequence
Sense(S)/
Disease
Mutation
(5' to 3')
Antisense(A)
MELAS A3243 TTACCGGGC
T
CTGCCATCT A
A3243G TTACCGGGC
C
CTGCCATCT A
MELAS T3271 ACTTAAAAC
T
TTACAGTCA S
T3271C ACTTAAAAC
C
TTACAGTCA S
LHON G3460 GAGTTTTATGG
C
GTCAGCGAA A
G3460A GAGTTTTATGG
T
GTCAGCGAA A
MERRF A8344 GAGGTGTTGG
T
TCTCTTAAT A
A8344G GAGGTGTTGG
C
TCTCTTAAT A
MERRF T8356 AACACCTCT
T
TACAGTGAA S
T8356C AACACCTCT
C
TACAGTGAA S
CARDIO- G8363 TTTACAGT
G
AAATGCCCC S
MYOPATHY G8363A TTTACAGT
A
AAATGCCCC S
NARP T8993 CAATAGCCC
T
GGCCGTACG S
T8993C CAATAGCCC
C
GGCCGTACG S
T8993G CAATAGCCC
G
GGCCGTACG S
LHON G11778 ATTATGATG
C
GACTGTGAG A
G11778A ATTATGATG
T
GACTGTGAG A
LHON G14459 ATAGCCATC
G
CTGTAGTATA S
G14459A ATAGCCATC
A
CTGTAGTAT S
LHON T14484 AGACAACCA
T
CATTCCC S
T14484C AGACAACCA
C
CATTCCC S
MELAS, mitochondrial encephalopathy, lactic acidosis, stroke-like events; LHON, Leber's
hereditary optic neuropathy; MERRF, myoclonic epilepsy and ragged red fibers; NARP, neur-
opathy, ataxia, retinitis pigmentosa.
6.
20X SSC: 3
M
NaCl and 0.3
M
trisodium citrate (Invitrogen, cat. no. 15557-044).
7.
Church wash buffer: 40 m
M
phosphate buffer, pH 7.2, and 1% SDS.
8.
10 mg/mL Sheared salmon sperm DNA.
9.
Agarose.
10.
Agarose gel electrophoresis buffer (1X TAE): 40 m
M
Tris-acetate and 1 m
M
EDTA,
pH 8.0, or 50X Tris Acetate-EDTA (Quality Biological, Inc., cat. no. 351-008-130).
11.
3X agarose gel loading dye: 0.125% bromophenol blue, 0.125% xylene cyanol,
and 7.5% of Ficoll 400.
12.
T4 polynucleotide kinase (10,000 U/mL) from New England Biolabs.
13.
T4 polynucleotide kinase buffer: 70 m
M
Tris-HCl, pH 7.6, 10 m
M
MgCl
2
, and
5 m
M
dithiothreitol (DTT).
14.
γ
-32P ATP (4500 Ci/mmol, 10
µ
Ci/
µ
L).
15.
Zeta-Probe GT membrane (Bio-Rad, cat. no. 162-0196).
