Myeloid Leukemia

Diagnosis of PML-RARA-Positive APL 117
Table 1
Primers for Reverse-Transcription Polymerase Chain
Reaction Analysis of PML-RARA Fusion Gene
Primer code Size Sequence (5'–3')
PML-A1 21 CAGTGTACGCCTTCTCCATCA
PML-A2 18 CTGCTGGAGGCTGTGGAC
RARA-B 20 GCTTGTAGATGCGGGGTAGA
PML-C1 21 TCAAGATGGAGTCTGAGGAGG
PML-C2 19 AGCGCGACTACGAGGAGAT
RARA-D 20 CTGCTGCTCTGGGTCTCAAT
Table 2
Size of Polymerase Chain Reaction (PCR) Products for Each of the PML-RARA
Primer Sets
First-round PCR Nested PCR
Primer sets PML A1–RARA B PML A2–RARA B PML C1–RARA D PML C2–RARA D
bcr 1 381 214
bcr 2 345* 178*
bcr 3 376 289
*The size of the bcr2 +ve products is variable as a result of variable breakpoint location
within exon 6 of the PML gene.
here described in detail. In dilution experiments, a sensitivity of 10 –2 to
10 –3 has been reached in first-round PCR, and 10 –3 to 10 –4 in nested PCR.
The sensitivity is at least one log better for bcr3 than for bcr1/2. Table 1 shows
the sequence of the primers, whereas Table 2 shows the size of PCR fragments
generated as a result of different breakpoint regions in the PML locus, and/or
the presence of alternative splicing of PML transcripts (4). In the case of bcr2
breakpoints, the sizes of PCR fragments are different due to the variability of
the breakpoint location within PML exon 6.
With respect to the original method we described (13), the Biomed-1 protocol
includes several advantages: (1) a common PCR protocol (annealing temperature,
MgCl 2 concentration, number of cycles, and so on) can be used for
the screening of different translocations in the same group of patients; (2) a
standardized method was validated for multicenter studies both for molecular
diagnosis and monitoring of leukemia patients. According to most investigators,
high-quality RNA and efficient RT are the crucial determinants for successful
RT-PCR of PML-RARA. Because of frequent leukopenia and the