Optimization On PCR And Genetic Instability Of DNA Repeat Sequence Associated With Human Genetic Diseases

It was found that expansions of simple DNA repeats were responsible for more than forty hereditary disorders in humans. One of common characteristics of the repeat sequences was dynamic mutation of non classical (mendelian) genetics when the mutant gene was transmitted from one generation to the next, and genetic anticipation was subsequently detected for this hereditary neurological disorders.As a basic method, PCR was widely used to detect the repeat sequence in recombinant plasmid when studying genetic instability associated with human genetic diseases. Four factors, including the concentration of template DNA, Taq DNA polymerase, primers and dNTP, were investigated to optimize the PCR reaction system of DNA repeat sequence by using orthogonal experiment method. The result indicated that the concentration of primers had great effect on the result of PCR amplification. Based on this result, the single factor experiment of the concentration of primer was carried out and the optimal concentration of the components in PCR 25μL reaction system, which includes 3 ng/μL template DNA, 0.75 U Taq DNA polymerase, 0.25 mmol/L dNTP and 0.3μmol/L primers ,was obtained. Comparing the results of optimized system with reference system, target product was increased obviously. The optimized system was the basis of the further investigation on genetic instability of DNA repeat sequences and molecular mechanism of this kind of genetic diseases.Massive PCR template was needed for detecting the changes of repeat sequence when we studied the genetic instability and influence of drugs. High speed centrifugation, TE boiling method and TE/SDS boiling method were used to treat E.coli in order to explore the rapid preparation method of PCR template with plasmid containing repeat sequence and the three methods could also get clear objective band. Comparing with each other, the results of centrifugating 5min, boiling 3min by TE and boiling 2.5min by TE/SDS were better effect. The three methods were rapid, simple, low cost , no pollution and simplification of preparation of PCR template, and it was basis of further study of rapid screening and identification of recombinant plasmid with repeat sequence.The repeat sequences may lead to the onset of genetic disorders when it exceed the threshold, so we could try to find a suitable way to inhibit the amplification of repeat sequences or delete the repeat sequences, and this had great practical significance to prevent and treat the genetic diseases. The plasmids containing repeat sequences (GAA)42 and (ATTCT)43 were treated for several times by MMC and EMS. It was found that two chemical drugs could result in the deletion of repeat sequences in different extent and the deletion rates of (GAA)42 were 86.7% and 43.3% after 9 times by MMC and 13 times by EMS, and the length of repeat sequence were in normal range in vivo for most of the experiments. The results showed that chemical drugs could promote deletion of long repeats associated human genetic disease, and it was the basis of further study of pathogenesis and treatment of these diseases.