| Simple sequence repeats(SSR), widely distributed in genomes of many organisms, closely relatedto molecular evolution, genic regulation and some genetic diseases, widely used in genetic marks andgenetic mapping, are catching people’s increasing attention by its biological significance. It wasreported that many SSR, including tetranucleotide repeats(TRs), can be elongated easily and abnormalextension of some can directly result in certain hereditary diseases. Hoever, the amplificationcharacteristics and mechanism of TRs is researched rarely. To systemly study on the amplification ofTRs,this research firstly choose representative20nt60TRs and6dinuleotide repeats(DRs) andcomprehensively explore their amplification features under the action of polymerases. It discusses theeffects of temperature, Tmof templates, time, sequence, concentration and the species of polymeraseson the amplification and the length of the products. Besides, the effects of partial palindrome in thesinge strand on the amplification and the amplification characteristics of some pentanucleotide repeats(PRs), hexanucleotide repeats (HxRs), heptanucleotide repeats(HpRs) and octanucleotide repeats(ORs)are also analysed. Lastly, basing on the revelation of inverted repeats (IRs) can raise the amplificationability of repetitive single strands, an program, written by Matlab, is made to find out the IR in thesequences. Then, genomes of some viruses, escherichia coli, saccharomyces cerevisiae and a little partof human genome are searched for IRs whose characteristics are analysed.The amplification results of single-strand TRs mainly includs following aspects. Most ofsingle-strand templates, even the sequences with no complementary bases inside like (AGGA)5, canbe elongated. The bands of products of nonpanlidromic templates are dispersive, but those ofpanlidromic templates are concentrated in some cases. Panlidromic single strands amplifies strongerthan nonpanlidromic ones, and the former own faster initial amplification rate, but the latter amplificatevery slow at first. DRS could be amplified at a broader range of temperature than TRS. Single strandswith more G and C were more suitable for amplification under higher temperature. Most strands whoserepetitive unit contains two same pyrimidines were amplified more easily than their complimentaryones. The concentration of products exhibited linear relationship with time at70℃. Nonpanlindromicsingle-strand TRs(ssTRs) are able to produce visible products in16h when its concentration is morethan10nM. However, panlidromic ssTRs can produce obvious products even at10pM. The roomtemperature polymerase Klenow fragment can not amplify ssTRs effectively and the thermophilicpolymerases’ ability depends on the sequence. The effiecnt amplification of TRs needs certain amountof polymerases and that of panlidromic TRs is lower. The amplification ability increases largely afternondromic ssTRs containing some complimentary sequences: when it locates near3’ end, short molecular can be easily produced and5’, the longer. The amplification results of single-strand PRs,HxRs, HpRs and ORs display that: the amplification ability of panlidromic templates is still strong andthat of most nonpanlidromic ones is weak; only one base difference in the repetitive unit betweennonpanlidromic and panlidromic templates can also lead to the weak amplification ability of former; thesame base variance in the different sequence can produce different effects. The results of restrictionendonuclease digestion indicated that the products had the same repetitive unit with their originalrepetitive sequences and are the simple elongation of the latter. Finally, an two-stage amplificationmodel, including amplification by intra-chain slide and mediated by hairpin-contained structure, wasproposed to provide information for the study of nonspecific amplification of repetitive sequences andpathogenetic mechanisms of relevant diseases.The amplification results of single-strand TRs mainly includs following contents. Nondromicdouble-strand DRs(dsDRs) amplify stronger than nondromic double-strand TRs(dsTRs) and the trendof the length of former’s products increasing with time is more clear. The repetitive double strandsamplify faster than the single strand which composed the them in short time (3h). The bands of dsTRs’products are also dispersive and the product length of some increases with time. The most suitabletemperature for amplification (MSTA) of repetitive double strand is close to their Tm, so templates withmore GC own higher MSTA. The G-C distribution in the double strands also influences theamplification ability. The templates with the same bases share the smiliar amplification features, suchas the similar varience of product concentration with time. The action of polymerase relies on thesequence and the activity of thermophilic polymerases is stronger than room temperature polymerase(Klenow fragment). The products are the tandem repetitive sequence whose repetitive unit is the sameas the templates.At last, an IR search is performed on genomes of some viruses, escherichia coli, saccharomycescerevisiae and a little part of human genome. The indicators of statistical analysis are the length ofsequence(L), the numer(N) and density (pN) of IRs, the number(mN) and ratio(rmN) of perfect IRs, theGap(G) of IRs, the arm length(A) of IRs, the match ratio of arm(rmA) of IRs and the ratio of A-T in thematch part(mrAT). The indicators of virus group vary in a big range: pN0.003-0.017, mN0.17-0.64, G22.0-42.2, A8.8-18.8and mrAT0.16-0.81. This reflects that, to adapt each own environment, differentviruses obtain the largely different genomes. The chromosomes of different escherichia coli strainsshare almost coincident indicatotrs: the relatively less pN(0.008), shorter arm(11.0) and lessmrAT(0.51). But the plasmids own bigger pN (0.01), rmN (0.48) and mrAT (0.55) than chromosomes.The chromosomes of saccharomyces cerevisiae share almost the same indicators, such as the big gap(37.0) and mrAT (0.72). The mitochondria of saccharomyces cerevisiae have the distinct features: thebig pN(0.04), which means there is1inverted repeat every25bp; The small rmN (0.14) and G (25.0);the arm is longer (33.1), but rmA is small (0.84); mrAT is bigger (0.94). Because of the randomness inchoosing the sequence for search, the indicators of segments of chromosomes range to some extents.The features of them can be summarized: bigger pN(0.01), less rmN(0.42), less G(34.7), a little lengerarm(>12), bigger mrAT(0.63). Specially, No.4, No.8, No.12, No.13, especially No.22chromosome segment owns many IRs whose arm are longer than200bp. Most of sequences for search own moreIRs than random one composed by the same bases, whereas some viruses tend to have less IRs. Thedistribution of gap of IRs in most sequences show the significant difference with that in random one,which is caused by the sum of IRs in them and also the outstanding traits of biological genomes.The First two experiments can provide information for the study of the amplificationcharacteristics and mechanisms of repetitive sequences in vitro. By searching IRs in some genomes, theresults can offer assistance for the study of sequence composition and the existing states of IRs inbiological genomes and unveil the secret of the abnormal amplification of some sequences in vivo. |
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