DYSTROPHIN基因第45~54外显子缺失连接片段的克隆和测序.pdf
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1、 ORIGINAL ARTICLE Cloning and sequencing of junction fragment with exons 45-54 deletion of dystrophin gene ZHONG Min,PAN Su-yue,LU Bing-xun,LI Wei (Department of Neurology,Nanfang Hospital,Southern Medical University,Guangzhou,Guangdong,510515 P . R . China) Corresponding author:PAN Su-yue,Email:pan
2、suyue82yahoo . com . cn 【Abstract】 ObjectiveTo study the mechanisms of dystrophin gene deletion,the junction fragment with exons 45-54 deletion were cloned and sequenced. MethodsA Duchenne muscular dystrophy(DMD)patient with exons 45-54 deletion has been substantiated by PCR amplification of the exo
3、ns. Then we used a PCR-based genome-walking method for localizing the breakpoints in introns 44 and 54. At last,the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoints in introns 45 and
4、 54. The sequencing result of the deletion-junction fragment was compared with the normal intronic sequences. ResultsA total of 2716 bp sequence containing the junction fragment was obtained. The 5 breakpoint was located in LINE/ L1 element of intron 44 and close to a matrix attachment region(MAR) .
5、 The 3 breakpoint was located in the mi- nor potential MAR with topoisomerasecleavage sites around. Beside the 3 breakpoint there was a 6 bp palindromic sequence. A 4 bp microhomologous sequence(AGAG)was in the joint of the deletion-junction fragment. Conclusion The nonhomologous recombination cause
6、d by L1 repeated element,topoisomerasecleavage sites,MARs and the non- homologous end joining of microhomologous sequence may be the important factors in this huge gene fragment deletion. 【Key words】 Duchenne muscular dystrophy; junction fragment; gene deletion INTRODUCTION Dystrophin gene mutation
7、is the molecular pathological ba- sis for pseudohypertrophic muscular dystrophy,a lethal X- linked recessive disease. Dystrophin gene mutation give pri- ority to deletion which accounts for 55%-65% in all the mutants. It is the principal method in exploring the mecha- nism of dystrophin gene deletio
8、n to know the position of local breakpoint of gene deletion and features of sequence contain- ing junction fragment by cloning and sequencing dystrophin gene deletion-junction fragment. At present the data of sequence containing dystrophin gene deletion-junction fragment are comparatively scarce. In
9、 order to further study the mechanism of dystrophin gene deletion,we use PCR to fulfill the cloning and sequencing of long fragment gene deletion-junction fragment in exons 45- 54,and give an analysis of breakpoint and gene deletion- junction fragment. MATERIALS AND METHODS Patient data A 9-year-old
10、 male Duchenne muscular dystrophy(DMD) patient without family history of DMD was substantiated with exons 45-54 deletion of dystrophin gene. Reagents Taq Plus DNA polymerase and dNTP were purchased from Shanghai Sangon Biological Engineering Technology & Ser- vice Co.,Ltd. LA Taq DNA polymerase in a
11、mplifying long fragment from TaKaRa Biotechnology(Dalian)Co.,Ltd. Agarose was imported from Biowest Agarose,Hispanagar Company,Spain. Other reagents were home made analyti- cally pure. Primer design This experiment involves 24 pairs of exon primers,of which 18 pairs are designed referring to literat
12、ure of Cham- berlain,et al . 1and Beggs,et al .2,while the other 6 pairs designed referring to literature of Abbs,et al . 3and Prior,et al . 4and obtained from Leiden Muscular Dystro- phy Pages(http: / / www.dmd.nl) . The experiment involves 27 pairs of intron primers consisting of 17 pairs of intro
13、n 44 and 10 pairs of intron 54. The design was fulfilled by primer on-line design program(http: / / frodo.wi.mit.edu/ cgi-bin/ primer3/ primer3-www. cgi) . The design principle of intron 44 forward primer is to design 5 pairs of primer,which di- vides the whole intron sequence into 6 tested regions
14、with the length about 40 kb roughly on average. One pair of primer was designed every 3 kb sequence in the confirmed break- point localized region. The design principle of intron 54 for- ward primer is to directly design 1 pair of primer every 3 kb sequence. Both primer designs are limited with thei
15、r PCR sequence length within 301-400 bp. In order to improve the specificity of cloned gene deletion-junction fragment,we re- designed 1 pair of matched-pair primer close to the break- point of intron 44 and intron 54(D5-F: 5- GTTATCTTATG- GATGCACGGTTTTG-3,D5-R:5-AGATTCTTCAGAGAT- CATGGATGGA-3) to be
16、 used in long fragment PCR amplifi- cation of joint fragment. All the primers mentioned above were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. Cloning of exons 45-54 deletion-junction fragment Genomic DNA of the DMD patient mentioned above was 831中华医学遗传学杂志 2006 年 4 月第 23 卷第 2 期Chin J
17、Med Genet,April 2006,Vol. 23,No. 2 extracted and prepared by routine phenol-chloroform meth- ods. The patient was confirmed to have exon deletion in exon 45 after primarily screening by PCR on 18 pairs of exon primers,and confirmed to have exons 45-54 deletion after adding PCR on exons 46,49,53-56 p
18、rimers. PCR condi- tion:at 96 for 2 min; at 94 for 30 s, annealing(the concrete temperature was determined by Tm value of each prime)for 30 s, at 72 for 1 min, 35 cycles; at 72 for 10 min. The appropriate position of the breakpoint in introns was found by using designed intron primers. If one pair o
19、f in- trons was amplificated into normal positive result while the other pair negative result in the neighboring 2 pairs of intron primers,we could judge the appropriate position of the breakpoint in introns. PCR condition is the same as above paragraph. Long fragment PCR amplification was directly
20、conducted on junction fragment of primers D5-F and D5-R. PCR con- dition:at 96 for 2 min; at 94 for 30 s, at 54 for 30 s, at 72 for 3 min, 35 cycles; at 72 for 10 min. Sequencing of gene deletion-junction fragment Sequencing after purifying successfully PCR production. The sequencing was performed b
21、y Shanghai Sangon Biologi- cal Engineering Technology & Service Co.,Ltd by de- terming double sequence of PCR production. Analysis of features of junction fragment and breakpoint sequence The determined sequence and normal introns sequence were compared to analyse the molecular features of 100 bp se
22、quence both sides of 3 breakpoint and 5 breakpoint. Analysis of intron sequence repeat was adopted by Repeat- Masker program(http: / / www. repeatmasker. org/ cgi-bin/ WEBRepeatMasker) ,matrix attachment regions( MARs) analysis by MAR-Wiz program (http: / / www.futuresoft.org/ MAR-Wiz) . RESULTS Pro
23、ved by PCR reaction of intron primer,5 breakpoint was appropriately located in about 178 kb site in intron 44, and 3 breakpoint was appropriately located in about 21 kb site in intron 54. The production fragment of about 2.7 kb in length was amplified from long fragment PCR reaction in gene deletion
24、-junction fragment(Fig.1) . A total of 2716 bp sequence containing the junction frag- ment was obtained,which was proved to be dystrophin gene fragment by comparing the similarity with http: / / www. ncbi.nlm. nih. gov/ genome/ seq/ HsBlast. htm with the total deletion length about 402 kb. The 5 bre
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- DYSTROPHIN 基因 45 54 外显子 缺失 连接 片段 克隆
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