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Duchenne/Becker muscular dystrophies (DMD/BMD) are allelic, X-linked recessive neuromuscular disorders that result from mutations in the human dystrophin gene[1]. The dystrophin gene is localized at the middle of the short arm of the X chromosome (Xp21)[2]. It has 79 exons and 78 introns spanning a physical distance of 2 500 kb and known as the largest gene of human beings. The human dystrophin gene is uniquely characterized by its high rate and different forms of mutation[3]. Analysis of patient DNA samples with dystrophin cDNA and genomic clones has revealed that 55% to 65% of all cases of DMD/BMD result from partial or all exon deletions, which is the major form of dystrophin gene mutation[4]. At present, multiple polymerase chain reaction (mPCR) has been widely used for detecting deletions of dystrophin gene[5-11]. In addition, multiplex ligation-dependent probe amplification (MLPA) array[11-14] and DNA microarray[15-17] are being developed.
In the present study, the 18 deletion-prone exons of dystrophin gene were cloned through a method of molecule clone with human genomic DNA as template. The cloned fragments were designed as probes for detection the major deletion-prone exons of dystrophin gene, to prepare for DNA microarray.

Dystrophin gene is actually the most complex human gene yet identified, which presumably accounts for its high rate and different forms of mutations and the high frequency of DMD/BMD in the human population. Deletion of part or all of the dystrophin gene is one of the major causes of DMD/BMD[20]. The dystrophin gene often splits in introns rich in AT bases, which results in deletion of one or several exons between break points[21]. It was early detected that dystrophin gene deletions cluster in two recombination hotspot regions, one proximal at the 5’ end of the gene, comprising exons 2-20 (30%), and the other at distal, comprising exons 44-53 (70%)[22]. The site and size of these deletions are very heterogeneous[23]. As a result of this clustering, the majority of deletions can be detected by examining only a subset of exons. In this study, the major 18 deletion-prone exons of dystrophin gene were cloned, which were in the above two hotspot regions.
It is the major method in the area of molecular biology and genetic engineering that target gene fragment is amplified by PCR and then PCR product is cloned[24]. In this study, the 18 primers designed by Chamberlain or Beggs were used in PCR to amplify target fragments. These primers do not contain restriction enzyme identifying sequences at their ends. pGEM-T Easy Vector contains a thymidine (T) in its multiple cloning region, and PCR product usually contains a adenosine (A) at its ends[25]. According to A-T, PCR product can be directly ligated with pGEM-T Easy Vector. It is more efficient and convenient[26]. In addition, pGEM-T Easy Vector multiple cloning region is flanked by recognition sites for restriction enzyme Not Ⅰ and there are no recognition sites for Not Ⅰ in these cloned fragments with the software for analysis of cutter, so that the inserts can be completely released only by Not I digestion. It is confirmed that the cloned fragments have high homology with dystrophin gene through sequencing and NCBI BLAST against GenBank.
These cloned fragments have been designed as oligo-nucleotide probes to make DNA microarray for the analysis of dystrophin gene deletions[27]. We temporarily name them “exon probes”. Actually, they are just the PCR products, not identified with the cDNA probes in Southern blot[28]. Our cloning of these fragments is of great importance as follows: Each fragment, amplified with a pair of primers targeted on one specific exon, is complementary to the specific exon. So it would show high specificity when hybridized to dystrophin gene. These cloned fragments are about 113-547 bp, which is the right physical length for immobilization on a solid support as probes to make microarray[29]. The specificity of hybridization can be guaranteed, and space influence on adjacent probes when hybridizing on solid support can be reduced efficiently[30]. These fragments have been cloned. In addition, the strains carrying recombinant plasmids which comprise different fragments have been reserved. The fragments can be produced massively, easily and economically by asexual reproduction of the strains. It will satisfy the demand of microarray for voluminous probes.

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