Enzymatic amplification of synthetic oligodeoxyribonucleotides: implications for triplet repeat expansions in the human genome.
The triplet repeat sequences (CGG)n, (GCT)n, and (CAG)n, which naturally occur in the human genome, can be autonomously expanded in human DNA by an as yet unknown mechanism. These in part excessive expansions have been causally related to human genetic diseases, the fragile X (Martin-Bell) syndrome, to myotonic dystrophy (Curschmann-Steinert), to spinal and bulbar muscular atrophy (Kennedy disease), and recently to Huntington disease. A GCC trinucleotide repeat was found to be expanded and methylated in the fragile site FRAXE on the human X chromosome. These findings were associated with mental retardation (Knight et al., 1993). In spinocerebellar ataxia type 1 (SCA1), a polymorphic CAG repeat was found to be unstable and expanded in individuals with that disease (Orr et al., 1993). We have demonstrated in in vitro experiments that the synthetic oligodeoxyribonucleotides (CGG)17, (CGG)12, (GCC)17, (CG)25, (CTG)17, or (CAG)17 plus (GTC)17, in the absence of added natural DNA, can be expanded with Taq polymerase in the polymerase chain reaction (PCR). Some expansion can already be detected after 4 PCR cycles. The E. coli Klenow DNA polymerase also functions in a similar amplification and expansion reaction performed at 37 degrees C without cycling. Other oligodeoxyribonucleotides, like, (CGG)7, (CGGT)13, or (TAA)17, are devoid of this property or have very low activity. The cytidine-methylated polymers (GCC)17 or (CG)25 yield expansion products of considerably reduced chain lengths. The expansion of the polymer (CGG)17 is affected by cytidine methylation to a lesser degree. A specific sequence and/or secondary structure and high CG content appear to be requirements for this expansion reaction by a possible slippage mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Behn-Krappa
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Institute of Genetics, University of Cologne, Germany.
Doerfler
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D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D008745:Methylation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D019914:Taq Polymerase
D009838:Oligodeoxyribonucleotides; D004247:DNA; D019914:Taq Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase