PCR METHODS
Ligation-mediated PCR for the analysis of atypical TCRD gene rearrangements
Ligation-mediated PCR (LM-PCR) was developed based on the principle of the Universal
GenomeWalker Kit (Clontech; Palo Alto, CA). Aliquots of 1 µg high molecular weight DNA
were digested with blunt end restriction enzymes (EcoRV, DraI, PvuII and StuI,) and 50 M
of an adaptor was ligated to both ends of the restriction fragments. The adaptor was prepared
by hybridization of two oligonucleotides: A-sense 5’-GTA ATA CGA CTC ACT ATA GGG
CAC GCG TGG TCG ACG GCC CGG GCT GGT and A-reverse 5’-ACC AGC CC-NH2.
The amine group blocks the extension of the 3’ end of the reverse oligonucleotide, thereby
preventing formation of an AP1 binding site on non-specific adaptor-ligated restriction
fragments and adaptor duplexes. The ligation products were subjected to two rounds of PCR
with nested adaptor-specific primers AP1 and AP2 and three sets of TCRD specific primers:
for the TRDJ1 region: J1(+102) and J1(+62), for the TRDD3 region: D3(+133) and D3(+77)
and for the TRDV1 region: V1(-275) and V1(-69). First round PCR of 50 µl total volume was
performed with: 1 µl of the ligation products, 10 pmol of the AP1 primer and the J1(+102),
D3(+133), or V1(-275) primer, 10 nmol each of dATP, dCTP, dGTP, dTTP (Applied
Biosystems, Foster City, CA), 1 µl Advantage Genomic Tth Polymerase Mix, and a Tth PCR
buffer (Clontech) including 1.1 mM Mg(OAc) 2 . After the initial denaturation at 94C for 3
min., 7 cycles consisting of 94C for 25 sec. and 72C for 3 min. followed by 32 cycles
consisting of 94C for 25 sec. and 67C for 3 min. were performed, with a final extension step
at 67C for 7 min. The second round PCR was performed with 1 µl of the first round PCR
product, 10 pmol of the AP2 primer and the J1(+62), D3(+77) or V1(-69) primer. Without
initial denaturation, 7 cycles consisting of 94C for 25 sec. and 72C for 3 min., followed by
32 cycles consisting of 94C for 25 sec. and 67C for 3 min. were performed and followed by
a final extension step at 67C for 7 min. The amplification products were analyzed in a 2%
agarose gel. The bands of the leukemic sample that differed in size from those in peripheral
blood of healthy individuals, used as a germline control, were excised from the gel and
purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and subjected to
direct nucleotide sequencing performed by a commercial sequencing facility (AGOWA;
Berlin, Germany).
RT-PCR and Competitive RT-PCR
Total RNA was isolated using RNAeasy columns as described by the manufacturer (Qiagen).
A total of 1 µg of RNA was converted to cDNA in a total volume of 20 µl containing RTbuffer (20 mM Tris; 25mM KCL), 1.25 mM MgCl2 (Applied Biosystems; Foster City, Ca,
USA), 5 mM DTT (Invitrogen; Groningen, The Netherlands), 5 µM random hexamers
(Amersham Pharmacia Biotech, UK), 1 U RNAsin (Promega Corp., Madison, WI, USA), 10
U Superscript RT enzyme (Applied Biosystems) and 1mM dNTPs (Amersham Pharmacia
Biotech, UK). RT-PCR of 50 µl total volume was performed with: 1 l cDNA, 10 pmol of
each primer, 10 nmol each dATP, dCTP, dGTP, dTTP (Applied Biosystems), 1.5 U
AmpliTaq Gold polymerase and a PCR Buffer (Applied Biosystems) including 10 mM TrisHCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2 and 0.001% (w/v) gelatine. The following primers
were used: 5’ primers: BCLf268 specific for the first exon of BCL11B, or BCLf624 specific
for the second exon; and 3’ primers: BCLr1029 specifc for the fourth exon of BCL11B,
C1(+96) specific for the first exon of the constant region of the TCRD locus and C4(+78)
specific for the fourth exon of the TRDC region. After the initial denaturation at 94C for 5
min., 40 cycles consisting of 94C for 1 min., 60C for 1 min. and 72C for 2 min. were
performed, followed by the final extension step at 72C for 5 min. Competitive RT-PCR was
performed under the same conditions using one 5’ primer: BCLf624 and two 3’ primers:
BCLr1029 and C1(+96). As controls brain tissue, 5 T-cell samples obtained from peripheral
blood of healthy individuals and the TALL-104 cell line (ATCC) were used.
Real-time quantitative PCR
Expression levels of wild-type BCL11B and the BCL11B-TCRDfusion mRNA were
determined by real-time quantitative PCR using the ABI PRISM 7700 Sequence Detector
TaqMan (Applied Biosystems). Two duplex vectors were constructed, containing a fragment
of the wild-type BCL11B or BCL11B-TCRD fusion product and, as a reference, a fragment of
the 2-microglobuline gene (2MG). The 2MG was cloned first in the T-A acceptor site and
subsequently the BCL11B or BCL11B-TCRD were cloned into the EcoRV restriction site of
the pCR2.1 TOPO TA Vector (Invitrogen). The vectors were linearized and the number of
copies was determined based on the DNA concentration, measured by spectrophotometry and
confirmed by a quantitative gel electrophoresis. Standard dilutions of the vector from 107 to
101 copies were prepared. In brief, PCR amplification of a total volume of 50 µl was
performed with 2 µl of cDNA, 25 pmol of each primers, 5 pmol of 6FAM-TAMRA probe and
the TaqMan PCR Core Reagent Kit (Applied Biosystems). The kit includes 1.5 U AmpliTaq
Gold polymerase, 4 mM MgCl2 PCR buffer with 10 nmol of ATP, CTP, GTP and UTP, and
0.5 U AmpErase Uracil N-glycosylase, to prevent a carry-over contamination. The Uracil Nglycosylase was activated by an initial incubation at 50ºC for 2 min., followed by
denaturation at 95C for 10 min to activate the AmpliTaq Gold polymerase. Subsequently 45
two-step cycles consisting of 95C for 30 sec and 62C for 1 min. in case of 2MG or 64C
for 1 min. in case of both BCL11B and BCL11B-TCRD, were performed. The BCL11B and
BCL11B-TCRDamplification products, obtained with the 5’ primer BCLf624 and the 3’
primers: BCLr1029 or C1(+96) respectively, were detected using a probe specific to the
second exon BCL11B. The 2MG reference was amplified using the 5’ primer 2MGf41,
located in exon 1 and the 3’ primer B2MGb372, located in exon 3, in combination with an
exon 2 specific probe.