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Amplification was initialized at an annealing temperature of 66°C with a
decrement of 0.5°C for every cycle for 45 s, using a thermal cycler (Primus 96;
MWG AG Biotech). Denaturation was performed at 94°C for 30 s and
extension at 72°C for 45 s. After an annealing temperature of 52°C was
reached in the 28th cycle, 12 additional cycles were performed at 52°C. The
PCR program for the first-round amplification of IGH rearrangements was the
same as that of the screening step. In every first-round PCR, H
2
O used for the
preparation of the PCR reaction was simultaneously amplified as a negative
control. For the analysis of bone marrow follow-up samples (CR) during the
course of therapy, we used 1
g of DNA for amplification. Ten
l of the PCR
products were electrophoresed on an agarose gel (FMC). For the following
ASO-PCR step, 10
l of the first PCR product were diluted in 990
lof5mM
Tris-HCl, 0.5 mM EDTA (pH 8.0).
ASO-PCR (Second-Round) on a Block Thermocycler. ASO primers
corresponding to each junctional region were designed according to the criteria
published previously (22). All ASO and PCR primers for this method were
obtained from BIG Biotech (Freiburg, Germany). For IGH analysis, the ASO
was derived from the third complementarity-determining region and was
always used as a reverse primer in combination with an inner forward primer
(Fig. 1). For the second-round PCR of IGH rearrangements, we designed
primers according to the recently published V
H
sequences (Ref. 24 and Table
2). For the analyses of the IGK, TCRG, and TCRD gene rearrangements, ASO
primers were used as either forward or reverse primers (Fig. 1). The sequences
of most of the inner primers have been reported previously (22). Some inner
primers have been newly designed: K9.5 (for Kde), 5⬘-GTT TAC AGA CAG
GTC CTC AG-3⬘;V
␦
1-33 (for V
␦
1), 5⬘-CCA GGG TTC TGA TGA ACA
GAA TGC-3⬘;V
␦
2-33 (for V
␦
2), 5⬘-TCA ACT GGT ACA GGA AGA CC-3⬘;
␦
11.5 (for D
␦
3), 5⬘-AAG CTG CTT GCT GTG TTT GTC-3⬘; and G9.5 (for
J
␥
1.3/2.3), 5⬘-CTA TGT TCT CTT TTA GTA TGA GC-3⬘.
PCR was performed on a block thermocycler (MWG Biotech) in a 50-
l
reaction mixture with 1⫻ PCR buffer, 1 unit of Taq polymerase recombinant
(Life Technologies), 1.5 mM MgCl
2
, 20 pmol of each primer, and 2
lofthe
diluted first-round PCR product. One reaction (referred as to H
2
O1), contain-
inga2-
l aliquot from the diluted first-round PCR reaction of H
2
O, and a
second reaction (H
2
O2), containing 2
lofH
2
O used for the second-round
PCR reaction, were included in every experiment. The PCR reactions were
incubated for 3 min at 94°C. The PCR condition consisted of 30 s at 94°C, 30 s
at 50–65°C, and 30 s at 72°C for 30 (IGH) or 25 cycles (IGK/TCRD/TCRG).
The annealing temperature was calculated based on the melting temperatures
of the two amplimers (Tables 2 and 3). Using a pause function of the
thermocycler, 20
l of the PCR reaction were taken from the PCR reaction five
cycles before the end of the program. A final elongation step of 7 min at 72°C
was performed.
PCR products at each cycle were electrophoresed on 2–3% agarose gels
(FMC). In cases where the sensitivity did not reach 10
⫺4
or a false-positive
product of BC DNA became visible, the experiment was repeated with differ-
ent numbers of PCR cycles. The detection limit of the ASO-PCR and the MRD
level of remission samples were determined by visual inspection and a den-
sitometric analysis with the 1-D scan software on the gel print workstation
(MWG).
ASO-PCR (Second-Round) on the LightCycler. A LightCycler DNA
Master SYBR Green I kit (Roche Molecular Biochemicals) was used to
perform ASO-PCR on the LightCycler for each rearrangement. The kit pro-
vides PCR-grade H
2
O, 25 mM MgCl
2
, and 10⫻ LightCycler DNA Master
SYBR Green I containing 10⫻ PCR buffer, a deoxynucleotide triphosphate
mixture (with dUTP instead of dTTP), 10 mM MgCl
2
, SYBR Green I dye, and
Taq DNA polymerase. One
l of the diluted PCR products from the first-round
PCR reaction was amplified in duplicate in a 20-
l reaction containing 2
lof
10⫻ LightCycler DNA Master SYBR Green I, 1.6
lof25mM MgCl
2
(final
concentration 3 mM; Roche Molecular Biochemicals), and 10 pmol of each
primer. Two negative controls (H
2
O1 and H
2
O2) were included in every PCR.
The running protocol was programmed on the LightCycler software, version
Table 2 Junctional regions, PCR primers, and ASO-PCR results of IGH analyses in B-precursor ALL
Patient
no. Rearrangement
a
Junctional region
b
Primers for ASO-PCR
c
Size
of PCR
product (bp)
Annealing
temp.
(°C)
Block thermocycler LightCycler
Detection
limit
(10)
n
Cycle
number
d
Melting
curve
e
Detection
limit (10)
n
Cycle
number
f
7V
H
3.7-J
H
2 ⫺1/tttcc/0/D
H
6-19/⫺4/cctactc/0 (F) CATAAAGCAAGATGGAAGTG 163 60 ⫺535B⫺6 25.0
(R) ACTGCTATACCCGGAAACTC
8V
H
3.30-J
H
6 ⫺14/aaggacccacacgggga/⫺7/D
H
3-3/⫺8/gg/⫺18
(F) TGGGTCCGCCAGGCTCCAGG 206 62 ⫺525C⫺5 19.0
(R) TCTCCCCGTGTGGGTCCT
11 V
H
3.43-J
H
6 ⫺5/gtaaatc/0/D
H
3-3
/⫺4/atactac/⫺18
(F) TGGGTCCGTCAAGCTCCGGG 201 50 ⫺530B⫺5 20.0
(R) TAATACGATTTACTTTGC
12 V
H
4.55P-J
H
5 ⫺1/ccccataggga/⫺10/D
H
3-22/0/ggg/⫺2
(F) TGAATCTGGGTCCGCCAGCC 214 64 ⫺530B⫺5 28.2
(R) CCACTACTATCTCCCTATGGG
13 V
H
3.11-J
H
6 ⫺3/tggggagg/⫺6/D
H
6-13/
⫺3/cgggggc/⫺3
(F) TGGATCCGCCAGGCTCCAGG 211 66 ⫺425B⫺6 17.5
(R) CGCCAGCTGCTGCTCCTCC
14 V
H
3.15-J
H
6 0/taccac/⫺5/D
H
2-2/⫺
3/tttgtgggccgtggagggggttaa/⫺3
(F) ACAACAGACTACGCTGCACC 148 62 ⫺530B⫺5 18.3
(R) GGTACTACTACAATGTGGTATC
22 V
H
3.66-J
H
6 ⫺11/ccgcataa/⫺8/D
H
2-2/
⫺10/ggctggg/⫺15
(F) TGGGTCCGCCAGGCTCCAGG 197 60 ⫺420A⫺4 13.1
(R) TGGTACTACTACTTATGCGG
23 V
H
1.69-J
H
4 ⫺1/cgg/⫺6/D
H
3-9/⫺6/
cccttacctccg/⫺4
(F) TGGGTGCGACAGGCCCCTGG 230 60 ⫺430A⫺6 19.6
(R) AGTCAACGGAGGTAAGGGAT
25 V
H
4.39-J
H
6 ⫺1/ggggaagtcctggggggag/⫺10/
D
H
6-13/0/gggttttc/0
(F) ATCTATTATAGTGGGAGCACC 177 60 ⫺525A⫺5 14.9
(R) AACCCGTACCAGCTGCCTCC
27 V
H
3.71P-J
H
4 0/gaatgaatctac/⫺10/D
H
6-6
/⫺2/actgggctcgtcg/0
(F) CAAAGCTAATGGTGGGACAAC 165 56 ⫺430B⫺4 14.3
(R) TACGAGCGTAGATTCATTC
35 V
H
3.21-J
H
5 ⫺1/gccccgcggctagtggattctacttt/⫺9 (F) GGGTCTCATCCATTAGTAGT 183 58 ⫺525B⫺5 15.2
(R) AAAGTAGAATCCACTAGCCGC
a
V
H
gene was determined according to the GenBank data (Accession No. AB019437; Ref. 24).
b
Lower case letters represent N-nucleotides. D
H
gene was determined according to the GenBank data (Accession No. X97051 S64822).
c
Underlined sequences correspond to N-nucleotides. F, forward; R, reverse.
d
The cycle number at which the detection limit was determined.
e
The melting curve patterns were classified as A, B, and C (see “Results”).
f
The crossing point with the X axis of the dilution step showing the detection limit.
3283
QUANTIFICATION OF MRD IN ALL PATIENTS BY LIGHTCYCLER
Research.
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