Leukemia (1998) 12, 976–981
 1998 Stockton Press All rights reserved 0887-6924/98 $12.00
http://www.stockton-press.co.uk/leu
BIOTECHNICAL METHODS SECTION (BTS)
BTS
Leukemia
Panhandle PCR: a technical advance to amplify MLL genomic translocation
breakpoints
CA Felix1 and DH Jones2
1
Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine,
Philadelphia, PA; and 2Department of Pediatrics, University of Iowa, Iowa City, IA, USA
Translocations involving a breakpoint cluster region of the MLL
gene at chromosome band 11q23 are the most common molecular abnormalities in acute leukemias of infants and acute
leukemias related to chemotherapy with DNA topoisomerase II
inhibitors. Molecular cloning of MLL genomic breakpoints by
PCR has previously been difficult because MLL has many
translocation partners and several breakpoints involve
unknown partner genes. We review a new approach to MLL
genomic breakpoint cloning called panhandle PCR. By adding
an oligonucleotide sequence to the unknown 3′ partner gene
that is complementary to a known 5′ MLL sequence, we have
been able to generate a genomic template with an intrastrand
loop for PCR schematically shaped like a pan with a handle.
The intrastrand loop contains the translocation breakpoint and
unknown partner DNA, while the handle contains the known 5′
sequence from MLL and a complement to that sequence. Primers both derived from MLL are used to amplify the breakpoint
by panhandle PCR. Panhandle PCR offers the advantage of
having specificity for the strand of interest at both primer
annealing sites without requiring specific primers for the many
partner genes of MLL. Panhandle PCR is a straightforward
method that represents a technical advance in MLL genomic
breakpoint cloning.
Keywords: MLL; translocation; breakpoint; partner gene; leukemia; PCR
Introduction
Translocations of an 8.3 kb breakpoint cluster region (bcr)
located between exons 5 and 11 of the MLL gene at chromosome band 11q23 are present in the majority of de novo leukemias of infants and in the majority of leukemias related to
chemotherapy with DNA topoisomerase II inhibitors.1–7 MLL
genomic breakpoint cloning may advance our understanding
of whether exogenous damage to the MLL gene by anticancer
drugs, environmental toxins or DNA topoisomerase II inhibitors in foods is the common factor in leukemia pathogenesis
in treatment-related and de novo cases.8–11 The potential role
of DNA topoisomerase II in the translocation mechanism and
the sufficiency of specific translocations for full leukemogenesis are different biologic questions that require isolation of the breakpoints.12,13 MLL gene rearrangements confer
Correspondence: CA Felix, Division of Oncology, Leonard and Madlyn Abramson Pediatric Research Center, Rm. 902B, The Children’s
Hospital of Philadelphia, 324 South 34th Street, Philadelphia, PA
19104-4318, USA; Fax: 215 590–3770
Received 20 May 1997; accepted 30 January 1998
a poor prognosis in infant ALL.3,4,14–17 MLL genomic breakpoint cloning also will determine whether rearrangements of the
MLL gene with different translocation partners affect prognosis further.
MLL genomic breakpoint cloning by PCR previously has
been difficult because many of the translocations are composed of known 5′ sequences from MLL, but 3′ sequences
from unknown partner genes. The MLL gene has an estimated
30 different partner genes.2 Although 11 partner genes of MLL
already have been cloned, specific sequence information is
often limited to cDNAs.18–28 Sequences of two-thirds of the
partner genes have not yet been determined and, thus, are not
available for PCR-based translocation breakpoint cloning. In
approximately one-third of cases with molecular MLL gene
rearrangement at the level of the Southern blot, karyotype
analysis does not detect the translocation or give information
about potential translocation partners.7 Other rearrangements
result from tandem duplication of several exons of the MLL
gene. The karyotypes in these cases do not show abnormalities at chromosome band 11q23.29,30 Thus, there is a need for
new technology to clone MLL genomic breakpoints directly
from genomic DNA.
We have used panhandle PCR, which amplifies genomic
DNA with known 5′ and unknown 3′ sequences from a template schematically shaped like a pan with a handle,31–33 as
a new approach to MLL genomic breakpoint cloning.34,35 The
adaptation of the method should amplify the breakpoints
within the bcr with unknown 3′ sequences.34,35
General description of panhandle PCR methodology
Identification of MLL genomic breakpoints within the
bcr
We isolate high molecular weight genomic DNAs for Southern blot analysis and panhandle PCR by ultracentrifugation
on 4M GITC-5.7M CsCl gradients.36 Since leukemia in infants
typically presents with high WBC and large tumor burden,
material for molecular analysis usually is plentiful. Before performing panhandle PCR, genomic DNA from the leukemia of
interest is examined for rearrangement of the 8.3 kb BamHI
fragment that encompasses the MLL genomic bcr by Southern
blot analysis.7 The sizes of the rearrangements on the Southern
blot are approximate sizes of the target sequences for PCR.34,35
Biotechnical methods section (BTS)
CA Felix and DH Jones
In treatment-related cases, material for molecular analysis
often is more limited. Additional breakpoint mapping by
Southern blot analysis, which consumes additional DNA, is
unnecessary either for design of primers or design of panhandle PCR conditions.
is homologous to exon 5 between the MLL sequence that is
complementary to the ligated oligonucleotide, and the translocation breakpoint. A nested PCR reaction with primers 3 and
4, also from MLL exon 5, enhances the yield of products from
panhandle PCR34,35 (Figure 1).
Formation of the template for panhandle PCR
Specific protocol to amplify MLL genomic breakpoint on
der(11) chromosome by panhandle PCR
The method summarized in Figure 1 amplifies the breakpoint
on the der(11) chromosome. The first step in making the template for panhandle PCR is restriction enzyme cleavage with
an enzyme that creates a 5′ overhang.31,33 For leukemias with
MLL gene translocations, BamHI is most appropriate because
virtually all MLL genomic breakpoints are within the same
8.3 kb BamHI restriction fragment.34,35 BamHI digestion produces a restriction fragment with known MLL sequence at the
5′ side of the translocation breakpoint and unknown 3′
sequence from the partner DNA.34,35 The DNA is treated with
calf intestinal alkaline phosphatase to prevent religation in
Step 2.31–33
The purpose of Steps 2 and 3 is to form the handle.31–33
Formation of the handle attaches known MLL DNA to 3′ of
the unknown partner DNA and brings the translocation breakpoint and unknown partner DNA within an intrastrand loop
or pan.34,35 Step 2 involves ligation of a single-stranded 5′
phosphorylated oligonucleotide to the 3′ ends of the digested
DNA. The 4-base 5′ end of the oligonucleotide is complementary to the 5′ overhang of BamHI-digested DNA; its 3′ end is
complementary to nucleotides in MLL exon 5, which is in the
5′ bcr.34,35 The sense strand (top strand in Step 2, Figure 1)
becomes the template strand in Step 3.
Formation of the handle is completed in Step 3 by intrastrand annealing of the ligated oligonucleotide to the complementary sequence in MLL and polymerase extension of the
recessed 3′ end.33–35 An aliquot of the BamHI-digested, ligated
DNA is added to a reaction mixture of DNA polymerase,
dNTPs, and PCR reaction buffer.33–35 We preheat reaction
mixtures to 80°C before addition of the DNA to prevent nonspecific annealing and polymerization. After addition of the
DNA, we heat reaction mixtures at 94°C × 1 min to make the
template single-stranded by heat denaturation. Intrastrand
annealing of the ligated oligonucleotide to its complementary
sequence in MLL and template-directed polymerase extension
of the recessed 3′ end occur during a 2 min ramp to 72°C
and incubation at 72°C × 30 s to complete formation of the
handle.33 The intrastrand loop contains the translocation
breakpoint and unknown partner DNA, while the handle contains known 5′ sequence from MLL and a complement to
that sequence.34,35
PCR amplification of MLL genomic breakpoints from
template DNA shaped like a pan with a handle
With MLL sequences at both ends of the template, we use
MLL primers all sense with respect to exon 5 to amplify the
breakpoint junction.34,35 The positions and orientations of the
primers with respect to the ligated oligonucleotide are shown
in Figure 1, Step 1. Step 4 is to add MLL primers and thermal
cycle. MLL primer 1 is homologous to exon 5 upstream to the
MLL sequence that is complementary to the ligated oligonucleotide, allowing primer 1 to anneal to the DNA previously
attached to the 3′ end of the ligated oligonucleotide by template-directed polymerase extension in Step 3. MLL primer 2
Step 1. BamHI digestion and calf intestinal alkaline
phosphatase treatment
(1) Digest 5 ␮g genomic DNA to completion with 40 U (8
U/␮g) BamHI (New England Biolabs, Beverly, MA, USA)
at 37°C for 2 h in the appropriate 1 × buffer containing
1 × bovine serum albumin (New England Biolabs) in a
reaction volume of 100 ␮l to create restriction fragments
with a 5′ overhang.
(2) To dephosphorylate the cleaved DNA, add 0.05 U of calf
intestinal alkaline phosphatase (Boehringer Mannheim
Biochemicals, Indianapolis, IN, USA). Prepare a 100 ␮l
stock of 0.01 U/␮l calf intestinal alkaline phosphatase by
diluting the 1 U/␮l calf intestinal alkaline phosphatase
supplied by the manufacturer 1:100 in 10 mM TrisHCl/1 mM EDTA (TE) buffer. Then add 5 ␮l of the
0.01 U/␮l calf intestinal alkaline phosphatase to the
100 ␮l digested DNAs and incubate the 105 ␮l reactions
at 37°C × 30 min.
(3) Purify the digested, phosphatase-treated DNA by glass
bead extraction using a GENECLEAN III kit exactly according to the manufacturer’s instructions for 5 ␮g genomic
DNA (BIO 101, La Jolla, CA, USA) in order to remove the
phosphatase. Elute the purified DNA in a final volume of
50 ␮l TE buffer. Reserve 25 ␮l at −20°C for later use as
the unligated control (c.f. Step 3, below).
Step 2. Ligation of single-stranded 5′ phosphorylated
oligonucleotide to the 3′ ends
The sequence of the 5′ phosphorylated oligonucleotide for
amplification of the translocation breakpoint on the der(11)
chromosome is 5′-GAT CGA AGC TGG AGT GGT GGC CTG
TTT GGA TTC AGG-3′. The 32-nucleotide 3′ end of the 5′
phosphorylated oligonucleotide is complementary to nucleotides 92–123 in MLL exon 5 in the 8.3 kb BamHI fragment
that defines the bcr. The 4-base 5′ end is complementary to
the 5′ overhang of BamHI-digested DNA and is designed
specifically to not reconstitute the BamHI site upon ligation,
as we added BamHI sites in PCR primers 3 and 4 to subclone
the products of panhandle PCR (c.f. Step 5, below).
(1) Resuspend the 5′ phosphorylated oligonucleotide in dH20
at a final concentration of 0.25 ␮g/␮l.
(2) Add reagents to generate a ligation mixture of a final 50 ␮l
volume: 16.9 ␮l dH20, 25 ␮l (2.5 ␮g) of digested, phosphatase-treated DNA, 2.1 ␮l (516 ng) of 5′ phosphorylated
oligonucleotide, 5 ␮l of 10 × ligase buffer, and 1 ␮l of 1
Weiss Unit/␮l T4 DNA ligase (Boehringer Mannheim
Biochemicals). Incubate overnight at 4°C. The 516 ng of
primer is in approximately 50-fold molar excess with
respect to the BamHI cleaved genomic DNA.33
(3) Purify the DNA again using the GENECLEAN III kit according to directions provided by the manufacturer (BIO 101).
Elute the ligated DNA in a final volume of 25 ␮l TE buffer.
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CA Felix and DH Jones
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CA Felix and DH Jones
Figure 1
Schematic of amplification of an MLL genomic translocation breakpoint by panhandle PCR. Step 1 in building the template
is digestion of genomic DNA with BamHI to produce a restriction
fragment with a 5′ overhang. The DNA has known MLL sequence at
the 5′ side of the translocation breakpoint flanked by unknown 3′
sequence from the partner DNA. Formation of the handle attaches
known MLL DNA to 3′ of the unknown partner sequence and brings
the breakpoint junction within an intrastrand loop or pan. First, a single-stranded 5′ phosphorylated oligonucleotide that is complementary
to a 5′ sequence in MLL exon 5, is ligated to the unknown 3′ end
(Step 2). The sense strand (top strand in Step 2) becomes the template
strand. Formation of the handle is completed in Step 3 by intrastrand
annealing of the ligated oligonucleotide to the complementary
sequence in MLL and polymerase extension of the recessed 3′ end.
With MLL sequences at both ends of the template, primers 1 and 2
from MLL exon 5 are used to amplify the breakpoint junction (Step
4). Nested PCR with primers 3 and 4 enhances yield (Step 5).
Step 3. Panhandle formation: addition of DNA to
Taq/dNTP mixture, denaturation, intrastrand annealing
and polymerase extension
(1) For each reaction, prepare 25 ␮l of 2 × PCR mix by adding
2.5 U (0.75 ␮l) Taq/Pwo DNA polymerase mix (Expand
Long Template PCR System; Boehringer Mannheim
Biochemicals), 0.7 ␮l of a 1:1:1:1 mixture containing
25 mM each dATP, dCTP, dGTP, dTTP, 5 ␮l of 10 × PCR
reaction buffer, and 18.55 ␮l of dH20. The 2 × mix may
be prepared as a bulk cocktail, pre-aliquoted and stored
at −20°C for future use.
(2) Add 18 ␮l of dH20 to 25 ␮l of 2 × PCR mix contained in
a 500 ␮l thin-wall tube (Perkin-Elmer, Norwalk, CT, USA)
and layer on 1 drop (苲50 ␮l) of mineral oil. To prevent
non-specific annealing and polymerization, preheat the
tube to 80°C in a thermal cycler.33
(3) Add a 200 ng aliquot (2 ␮l) of the digested, ligated DNA
to the pre-heated reaction mixture. After addition of the
DNA, the reaction mixture will contain 2.5 U Taq/Pwo
DNA polymerase mix (Expand Long Template PCR System; Boehringer Mannheim Biochemicals), 385 ␮M each
dNTP (Expand Long Template PCR System; Boehringer
Mannheim Biochemicals), and PCR reaction buffer at
1.1 × final concentration in a 45 ␮l volume. Heat the reaction mixture at 94°C × 1 min to make the template singlestranded.33 Include a negative control reaction containing
200 ng (2 ␮l) of the digested, phosphatase-treated DNA
that was not ligated (c.f. Step 1) and a reagent control reaction without DNA.
(4) For intrastrand annealing of the ligated oligonucleotide to
the complementary sequence in MLL and polymerase
extension of the recessed 3′ end, follow the 94°C heat
denaturation with a 2 min ramp to 72°C and incubation
at 72°C × 30 s.
(5) Follow with an 80°C soak file. Maintaining the tube at
80°C before addition of the PCR primers in Step 4 prevents
priming at low stringency and the generation of nonspecific products, and thus provides a hot-start for the initial
PCR.37
Step 4. Addition of MLL primers 1 and 2 and thermal
cycling
(1) The sequence of MLL primer 1 is 5′-TCC TCC ACG AAA
GCC CGT CGA G-3′ and the sequence of MLL primer 2
is 5′-TCA AGC AGG TCT CCC AGC CAG CAC-3′. With
the reaction mixtures at 80°C, add 12.5 pmoles each
primer in 2.5 ␮l volumes to below the mineral oil layer.
This will bring concentrations in 50 ␮l final reaction volumes to 350 ␮M each dNTP and 1 × PCR reaction buffer.
In the original method, primer 2 had a nucleotide added
to its 5′ end that was not complementary to the template.
This was a precaution to prevent short-circuiting of the
reaction when using Taq DNA polymerase alone during
PCR, since short-circuiting could occur by the annealing
of the 3′ end of one strand of a short nonspecific PCR
product to the template DNA. The necessity of this precaution was not tested and the success of the method
when using long-range PCR reagents that include a DNA
polymerase with 3′ exonuclease activity suggests that the
precaution is unnecessary.
(2) If Southern blot information is available, the information
can be used to determine the duration of the elongation
segment in the PCR reaction (苲1 min/kb).34,35 To amplify
products 8.3 kb and 7 kb in size, we used the following
conditions: initial denaturation at 94°C × 1 min; 10 cycles
at 94°C × 10 s, 68°C × 7 min; 20 cycles at 94°C × 10 s,
68°C × 7 min (increment 20 s/cycle); final elongation at
68°C × 7 min.34 Shorter products can be amplified using
shorter elongation times.35
Step 5. Perform nested PCR using primers 3 and 4
(1) Figure 1 shows positions of the nested primers 3 and 4.
The sequences are 5′-A GCT GGA TCC GGA AAA GAG
TGA AGA AGG GAA TGT CTC GG-3′ and 5′-A GCT GGA
TCC GTG GTC ATC CCG CCT CAG CCA C-3′, respectively. Underlined sequences are BamHI sites. To 25 ␮l of
2 × PCR mix (c.f. Step 3), add 19 ␮l of dH2O, 2.5 ␮l each
(12.5 pmoles) of primers 3 and 4, and a 1 ␮l aliquot of
the initial PCR reaction products. Layer with mineral oil.
Use the same conditions for nested PCR as for the initial
panhandle PCR reaction.
Visualize 3 ␮l of the nested panhandle PCR reaction
products on an ethidium-stained minigel. Detection of a
product of the same size as the BamHI fragment on the
genomic Southern blot will be the first indication that the
amplified products at the end of Step 5 contain the target sequence.
Subcloning and sequencing of the products of
panhandle PCR
Primers 3 and 4 for the nested panhandle PCR reaction contain BamHI restriction sites for subcloning. The products of
panhandle PCR are agarose gel-isolated and subcloned for
sequencing of the translocation breakpoint and the unknown
partner DNA.
To validate the results, we routinely have designed primers
encompassing the translocation breakpoint from sequences of
the subcloned products of panhandle PCR, amplified fresh aliquots of genomic DNA from the leukemia, and performed
direct genomic sequencing.
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CA Felix and DH Jones
980
Guidelines for troubleshooting
One problem that may be encountered is the generation of
nonspecific products. The hot-start at 80°C at the first PCR
step prevents nonspecific priming and the generation of such
products.37 However, if multiple bands are seen following the
nested PCR amplification, raising the annealing temperature
by 1°C or 2°C in the nested PCR reaction may eliminate
the shorter products. Alternatively, reducing the amount of
DNA polymerase also may be helpful if this problem is
encountered.
translocation breakpoints where the partner gene is undetermined.
Acknowledgements
CAF was supported by National Institutes of Health Grant
No. 1R29CA66140–02, American Cancer Society Grant
No. DHP143, Leukemia Society of America Scholar Award
(1996–2001), Children’s Cancer Group, National Leukemia
Research Association Grant in Memory of Maria Bernabe Garcia, The Children’s Hospital of Philadelphia High Risk High
Impact Grant.
Discussion
References
Panhandle PCR was developed to clone genomic DNA with
known 5′ sequence flanked by unknown 3′ sequence. Panhandle PCR previously amplified target sequences 2–4.4 kb in
size from human ␤ globin and cystic fibrosis transmembrane
conductance regulator (CFTR) test genes.31,32 The MLL bcr lies
within an 8.3 kb region between exons 5 and 11, a length
suitable for PCR analysis. The adaptation of panhandle PCR
for MLL genomic breakpoint cloning ligates a phosphorylated
oligonucleotide of known sequence complementary to a
region in MLL exon 5, to 3′ sequence from the unknown partner gene. We have already used the method to clone three
MLL genomic breakpoints and have amplified target
sequences 2.5 kb to 8.3 kb in size.34,35 Successful application
of the method in a case of infant ALL and in two treatmentrelated leukemias identified the MLL genomic breakpoints and
previously uncharacterized intronic sequences in the partner
genes. In two of the three cases, the karyotype did not reveal
the translocation partner.34,35
Panhandle PCR offers advantages over other strategies for
genomic translocation breakpoint cloning. The method is considerably less labor-intensive than conventional genomic
cloning using ␭ phage, the method by which several MLL genomic breakpoints were isolated early on.38–41 Unlike onesided anchored PCR, panhandle PCR has specificity for the
strand of interest at two primer annealing sites.42 Unlike longdistance PCR,43,44 panhandle PCR does not require specific
primers for the many partner genes of MLL. Inverse PCR has
also been used to amplify a carcinogen-induced thymidine
kinase gene rearrangement.45 The approach included restriction enzyme cleavage on each side of the rearrangement, circularization of the restriction fragment with T4 DNA ligase,
and PCR amplification with outward primers both derived
from the known side of the rearrangement.45 However, successful amplifications were limited to template sizes ⬍1 kb.45
We believe that panhandle PCR is a technical advance
towards understanding the molecular pathogenesis of leukemias with MLL gene translocations. Although panhandle PCR
contains several steps including multiple glass-bead extractions, the initial success demonstrates that panhandle PCR is
a straightforward method to clone MLL genomic breakpoints.34,35 The maximum length of the target sequence that
can be amplified by this method remains to be determined,
but the same phosphorylated oligonucleotide and PCR primers from MLL exon 5 should be suitable for amplifying
additional MLL genomic breakpoints contained on BamHI
restriction fragments up to at least 8.3 kb.34,35 The method is
practical in cases where genomic DNAs are limited. Panhandle PCR is a definitive PCR approach for identifying
additional new partner genes of MLL and amplifying other
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