Amplification and sequencing of genomic breakpoints located within the M-bcr region by Vectorette-mediated polymerase chain reaction.
The polymerase chain reaction (PCR) cannot be used to amplify the breakpoint in the chimaeric BCR-ABL gene in CML and acute leukaemias due to the large variation in the sites of breakpoint in the BCR gene (within a 5.8 kb region) and in the ABL gene (within a 150 kb region). The disease state is usually monitored using RNA-PCR to monitor abnormal transcripts. We have used a new modification of the PCR to amplify breakpoints within zone 3 of the M-bcr. A synthetic oligonucleotide linker, the Vectorette, is ligated to restriction digested DNA, and amplification is carried out between primers for a known target sequence and the Vectorette linker. Three Philadelphia chromosome Ph1-positive CML patients with breakpoints within the ALU region of zone 3 have been amplified and the sequence immediately around the breakpoint determined. The breaks occurred within 70 bp and two were only 14 bp apart. The Vectorette-PCR technique has the potential to rapidly identify and sequence breakpoints, and will enable the design of patient-specific primers to monitor disease progression, particularly following bone marrow transplantation.
Mills
K I
KI
Department of Haematology, Glasgow Royal Infirmary, Scotland, UK.
Sproul
A M
AM
Ogilvie
D
D
Elvin
P
P
Leibowitz
D
D
Burnett
A K
AK
D001483:Base Sequence; D004262:DNA Restriction Enzymes; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D016313:Genes, abl; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*
D009841:Oligonucleotides; D016044:Fusion Proteins, bcr-abl; D004262:DNA Restriction Enzymes