A new fingerprint method for sequence analysis of chromosomal translocations at the genomic DNA level.
Chromosomal rearrangements constitute a significant feature of leukemogenesis and malignant transformation in general. Nucleotide patterns in the immediate vicinity of the break point may provide important information about the underlying causalities, eg illegitimate recombination events mediated by topoisomerase II, Alu repeats, or VDJ recombinase. In order to facilitate the determination of those DNA patterns, we developed a new fingerprint approach. In a first step, two DNA fragments were independently amplified by long distance PCR: the genomic region carrying the break point and the normal nonrearranged counterpart. Subsequently, both PCR products were digested with restriction enzymes, end-labelled with a fluorescent dye, and subjected to high resolution polyacrylamide gel electrophoresis. By comparing the restriction patterns of the rearranged and the nonrearranged PCR fragments, the break points could be easily localized within a size range coverable by a single sequencing reaction. Finally, the exact DNA sequence across the break point was directly determined. The 'fingerprint' technique is fast, reliable and enables the assay of multiple samples in parallel.
Leis
T
T
Department of Pediatrics, Hematology, and Oncology, Children's Hospital, Justus-Liebig University, Giessen, Germany.
Repp
R
R
Borkhardt
A
A
Metzler
M
M
Schläger
F
F
Harbott
J
J
Lampert
F
F
D001483:Base Sequence; D002880:Chromosomes, Human, Pair 11; D002894:Chromosomes, Human, Pair 4; D002899:Chromosomes, Human, Pair 9; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D016172:DNA Fingerprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics; D015321:Gene Rearrangement; D015894:Genome, Human; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D008969:Molecular Sequence Data; D051788:Myeloid-Lymphoid Leukemia Protein; D016133:Polymerase Chain Reaction; D011519:Proto-Oncogenes; D014157:Transcription Factors; D014178:Translocation, Genetic
D004268:DNA-Binding Proteins; C497422:KMT2A protein, human; D014157:Transcription Factors; D051788:Myeloid-Lymphoid Leukemia Protein; D004247:DNA; D011495:Histone-Lysine N-Methyltransferase