Amplification of GAA/TTC triplet repeat in vitro: preferential expansion of (TTC)n strand.
Several human hereditary neuromuscular and neurodegenerative diseases are caused by abnormal expansion of triplet repeat sequences (TRSs) CAG/CTG, CGG/CCG, or GAA/TTC on certain chromosomes. It is generally accepted that multiple slippage synthesis accounts for the instabilities of TRS. Earlier in vitro experiments by Behn-Krappa and Doerfler showed that TRS with high GC content can be expanded. In contrast, here we demonstrated that certain AT-rich TRSs, (TTC)17, (GAA)10/(TTC)10 and (GAA)17/(TTC)17, were also expansion-prone in PCR. With respect to the sequence of TRS, surprisingly, we found that the AT-rich (GAA)17/(TTC)17 extended more efficiently than the GC-rich (CAG)17/(CTG)17. This strongly suggested that the AT content of the repeat may influence TRS expansion. Furthermore, to examine the expansion of single-stranded TRS, we showed that only (TTC)17, but not the complementary (GAA)17, can be expanded. This suggested that a T-T mismatch may stabilize compatible secondary structures, most likely hairpins, for slippage synthesis. However, another poly-pyrimidine TRS, (CCT)17, is not amplification-prone in PCR. Due to the high C-content, this TRS is unlikely to adopt hairpin structures at the high pH used for PCR. Thus, the single-stranded PCR experiment may serve as an indirect assay for the ability of a sequence to adopt a hairpin conformation. When amplification was performed in reactions using Klenow DNA polymerase, only the double-stranded TRSs can be expanded. The reaction rate for (GAA)10/(TTC)10 was slower than for (GAA)17/(TTC)17, suggesting that the length of the repeat may be important for the amplification of TRS. The findings of these in vitro experiments may aid in understanding TRS expansion in vivo.
Department of Food Health, Chia-Nan College of Pharmacy and Science, Tainan 717, Taiwan. imwu@chna.edu.tw
M J
MJ
Wu
L W
LW
Chow
M
M
Hsieh
D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D030342:Genetic Diseases, Inborn / Q000235:genetics; D006801:Humans; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013696:Temperature; D018911:Trinucleotide Repeats / Q000235:genetics*
D017931:DNA Primers; D004256:DNA Polymerase I