| Duchenne and Becker muscular dystrophies (DMD/BMD) are allelic, X-linked recessive neuromuscular disorders that result from mutations in the human dystrophin gene. They are characterized by muscular atrophy and atony. It is reported that approximately 1/3500 newborn males is affected by DMD/BMD. Because of their high incidence and serious endangerment, they have become the focus of researches on hereditary diseases. There is no effective therapy for them, so it is important to find patients and carriers, and to do pregnant diagnoses.It is confirmed that the dystrophin gene is localized to the middle of the short arm of the X chromosome (Xp21). It has 79 exons and 78 introns spanning a physical distance of 2500kb making it the largest known gene of human beings. Its coding product is named dystrophin, with 427kD, locating in membrane of muscular cells. It is be of very important to maintain the normal function of muscular cells. There are high rate and different forms of mutations in the dystrophin gene. Early researches found that one third of all these cases arise from a new mutation. Analysis of patient DNA samples with dystrophin cDNA and genomic clones has revealed that 55%-65% of all cases of DMD/BMD result from partial gene deletions, about 25% from pouit mutations, 5%-10% from partial gene duplications, and the remainer from mini-deletions and mini-duplications. Deletion of part or whole of thedystrophin gene is one of the major causes of DMD/BMD. The dystrophin gene often splits in introns rich in AT bases, which results to deletion of one or several exons between break points. It was early observed that dystrophin gene deletions cluster hi two recombination hotspot regions, one proximal at the 5' end of the gene, comprising exons 2-20 (30%), and the other more distal, comprising exons 44-53 (70%). The site and size of these deletions are very heterogeneous. As a result of this clustering, the major deletions can be detected by examining a subset of exons.In recent years, genetic research on DMD/BMD has made considerable progress. Gene diagnoses for DMD/BMD are available in many hospitals, such as Southern blotting with cDNA probes and polymerase chain reaction (PCR) with several sets of primers. While the current diagnostic methods are extremely useful, we find they suffer from several limitations in our practice. The main is their low efficiency. DNA microarray technique can be used for detection of all exons of dystrophin gene and is more efficient.Our experiment included foundational and clinical researches on DNA microarray technique to dystrophin gene deletion detection. Foundational research was about preparation of exon probrs via molecular cloning and preparation of DNA microarray via printing after syntheses. Clinical research was about analyses of dystrophin gene deletions in DMD/BMD patients via DNA microarray technique. The methods and results are shown as follow:1. Human genomic DNA was prepared from whole-blood leukocytes of healthy people. Eighteen fragments of dystrophin gene were obtained through PCR amplification with human genomic DNA as template and the eighteen pairs of primers respectively. The fragments was extracted from 2% agarose gel, and then connected with pGEM*-T Easy vector. The recombinants were transformed into E.coli JM109 competent cells, followed by being planted on LB/Amp/IPTG/X-Gal plates and being cultured. Select positive transformants with blue/white color screening, then extract recombinant plasmids DNA and digest them with restriction enzyme Not I. DNA sequences of the fragments were analyzed on an automatic DNA sequencer. Nucleotide analyses were performed through National Center for Biotechnology Information (NCBI) BLAST version 2.0 against the GenBank. The results shown that these amplified fragments were Exon 4, 44, 12, 8, 51,17, 19, 48, 45, 52, 60, 47, 6, 13, 50, 43, 3 and 49 of dystrophin gene respectively, with the high homology with dystrophin gene searched in GenBank. These fragments are 196bp, 268bp, 331bp, 360bp, 38...
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