Myeloid Leukemia

Detection of BCR-ABL Mutations 97
2.2. Sequencing Reaction
1. Sterile 0.2-mL capped PCR tubes (Interpath Services).
2. Sterile 1.5-mL capped PCR tubes (Interpath Services).
3. ABI Prism Dye terminator cycle sequencing ready reaction kit (Big Dye 3) (Applied
Biosystems). Aliquot into 30-µL lots and wrap in foil to protect from the
light. Store at –20°C.
4. PCR primers. All primers are ordered as a 50 µM solution and stored in 30-µL
lots at –20°C.
5. 75% isopropanol that is prepared at least weekly.
6. Glycogen, molecular-biology grade (Roche Diagnostics).
7. Mutation Surveyor software (SoftGenetics).
3. Methods
3.1. Semi-Nested PCR Reaction
Full details of methods for peripheral blood or bone marrow sample preparation,
RNA extraction, cDNA synthesis, and real-time quantitative PCR analysis
of BCR-ABL and BCR appear in Chapter 4. These procedures are performed
prior to mutation analysis and determine the quality and suitability of the
sample (see Note 1).
1. Primers were designed using Primer Express software. The sequence of the primers
for CML and ALL patients with the b2a2 or b3a2 BCR-ABL transcripts are:
First-step forward primer: 5� tga cca act cgt gtg tga aac tc
First-step reverse primer: 5� tcc act tcg tct gag ata ctg gat t
Second-step forward primer: 5� cgc aac aag ccc act gtc t
The second-step reverse primer is the same reverse primer that is used for the
first-step PCR. The first-step forward primer hybridizes in BCR exon b2 (b2 may
alternatively be referred to as BCR exon 13).
In the case of ALL patients who have a BCR-ABL transcript that involves the
fusion of BCR exon 1 to ABL exon 2 (e1a2 BCR-ABL transcript), an alternative
first-step forward primer is used that hybridizes to BCR exon 1:
e1a2 transcript: First-step forward primer: 5� acc gca tgt tcc ggg aca aaa g
The reverse primer and the second-step forward primer are the same primers that
are listed above for b2a2 and b3a2 transcripts.
2. Thaw the PCR reagents and first-step primers and mix thoroughly before use
(except for the enzyme, which is stored at –20°C until added to the master mix).
Centrifuge briefly to collect the material at the bottom of the tube.
3. Prepare a master mix on ice. The amount of master mix prepared is dependent on
the level of BCR-ABL transcripts, and two separate master mixes may be required
(see Note 1). For samples with low BCR-ABL levels that require the addition of 3 µL
of cDNA (150 ng), pipet the following into a 1.5-mL tube for each patient sample
+ 1 positive control + 1 no-template control + 1: 0.75 µL MgCl 2, 1.25 µL dNTP
(15 mM mix), 2.5 µL 10X Expand long template Buffer 3, 0.15 µL of the forward