Expansion Of Trinucleotide Repetitive Sequences And Its Mechanism

The widely present simple repetitive sequences in genomes are significant forstudying molecular evolution, genetic diversity, and some hereditary diseases. Thetrinucleotide repetitive sequences are researched better among these repeats, but mostof them were just been treated as an unusual phenomenon or aimed at some geneticdiseases, which were scattered and the mechanism were too superficial to bepersuasive. All of the64kinds of18bp trinucleotide repetitive sequences and the32kinds of complementary strands were chosen to study the amplification with thecatalysis of DNA polymerase under isothermal condition. Some of the influencingfactor like reaction temperature, sequences, the type and concentration of DNApolymerase to the amplification efficiency and length of the products were researchedcomprehensively. The result showed that trinucleotide repetitive sequences have theuniversal character of amplification. The expansion of single strands andcomplementary strands were different. In addition, the inbibitional effect of theforeign genes to simple sequence repeats was also discussed.All of the trinucleotede repetitive sequences could expand to long DNA besidesthe repeats with the single base composition, and the variance in sequence had greateffect on the amplification efficiency. The repeats with more GC, especially thosewith G and C in one of the strands were much easier to grow. This kind of expansionwas so quick that the products more than dozens of kb could be detected just in1h to4h. The extension products could be completely digested by restriction endonucleasewith special recognition sites. It showed that the products contain lots of short repeats.Products were sequenced to confirm that the product is indeed composed of a largenumber of trinucleotide repeats itself and complementary repeats. With the lowconcentration of5U/mL of Vent（exo-） DNA polymerase and a low concentration of1nM of short repeats, the expansion capability were still very strong, whichshowed the expandable feature of short repeats. On the basis of above research, thepossible expansion mechanism was proposed.For the thermostatic expansion of duplexes, Agarose and polyacrylamide gelelectrophoresis showed that the product has narrow size distribution of molecules.Take （AAA）₆/（TTT）₆for an example, the expansion has a linear dependence of thelength of products on the reaction time within limits. The sequences with1/3GCcontent had higher expansion rates. The elongation products more than2.0kb couldbe obtained within4h, with the expansion rate of8.0kb/min. The expansion becameweakened when the products were increased to certain extent to be saturated. Thetemperature had great impact on the expansion, and the short repeats could becatalyzed by DNA polymerase at37℃. With the increase of reaction temperature, thedifference of the products became obvious. The digestion of the products showed thatthey were composed of a large number of repeat itself and complementary repeatsunits. Finally, the mechanism of double-stranded repeats, which is called "replicationslippage" in our study was proposed. It was expected to lay the foundation for furtherstudy on molecular evolution of repeats and genetic test of nonspecific amplification.Studying the interaction between short repeats and non-repetitive sequences, wefound that the exogenous non-repetitive sequence has inbibitional effect on expansionof repeats. The addition of100nM non-repetitive DNA could inhibit the abnormalexpansion completely. This kind of inhibition got released when adding more DNApolymerase, which showed that the exogenous non-repetitive sequences and therepeats were in the competitive relation in combining DNA polymerase. Theexogenous DNA got priority, leading to the expansion of the repeats became weakeven though they have some kind of expandable structure. It can be inferred that theextension of the repeat sequence is a kind of non-specific extensions. This researchprovided a reference for further studies to inhibit non-specific expansion of therepeats.