基于改进的重叠延伸聚合酶链式反应技术快速制备DNA分子质量标准
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摘要
采用改进的重叠延伸聚合酶链式反应(polymerase chain reaction, PCR)技术,简便、快速地制备DNA分子质量标准。首先,利用普通PCR技术扩增5'和3'两端序列一致的DNA片段。随后,在重叠延伸PCR的变性和退火过程中,2个单链的3'端互补序列可形成双链,且这些单链片段可同时作为模板和引物,在PCR延伸过程中延长片段。随着反应循环数的增加,小片段产物逐渐减少,大片段产物逐渐增多。取不同循环数的重叠延伸PCR产物进行琼脂糖凝胶电泳检测,根据电泳结果选择最佳的PCR循环数或不同循环数的最佳组合,无须回收纯化,即可得到分子质量相差100 bp的DNA分子质量标准。经琼脂糖凝胶电泳检测,所制备的DNA分子质量标准条带清晰、分子质量准确、条带均一性好,可用于标记DNA分子质量大小。总之,本研究用于DNA分子质量标准制备的方法操作简单,周期短,成本低,无副产物,且能够根据具体需求定制DNA分子质量标准的大小。
DNA marker is a set of standards that are used to indicate the approximate molecular-mass size of certain DNA fragment which separated by agarose gel electrophoresis. In the present study, a straightforward method for preparing a user-customizable DNA marker by an improved overlap extension polymerase chain reaction(PCR) was established. The DNA fragment, containing the identical sequence in its 5' and 3' ends, was amplified by a conventional PCR. In the following denaturation and annealing process performed by the overlap extension PCR,two single-stranded DNA in the 3' end of which had complementary base sequences could form double-stranded DNA. In the extension process, these single-stranded DNA was utilized as both template and primer. The number of small fragments of DNA decreased and the number of large fragments of DNA increased gradually with the increased number of PCR cycles. The products of different cycles of overlap extension PCR were taken for agarose gel electrophoresis. The optimal PCR cycle or optimal combination of different PCR cycles was chosen according to the results of electrophoresis. Then 100 bp DNA ladder marker was obtained by the overlap extension PCR under the chosen condition. There was no need for purification of PCR products as the amplified fragments could be directly used in the agarose gel electrophoresis. The bands of prepared DNA marker were clear and accurate, and could be used for molecular studies. In sum, this method for DNA marker production is simple, time saving, cost effective, byproduct free and user-customizable in comparison with current ones widely used in most laboratories.
DNA marker is a set of standards that are used to indicate the approximate molecular-mass size of certain DNA fragment which separated by agarose gel electrophoresis. In the present study, a straightforward method for preparing a user-customizable DNA marker by an improved overlap extension polymerase chain reaction(PCR) was established. The DNA fragment, containing the identical sequence in its 5' and 3' ends, was amplified by a conventional PCR. In the following denaturation and annealing process performed by the overlap extension PCR,two single-stranded DNA in the 3' end of which had complementary base sequences could form double-stranded DNA. In the extension process, these single-stranded DNA was utilized as both template and primer. The number of small fragments of DNA decreased and the number of large fragments of DNA increased gradually with the increased number of PCR cycles. The products of different cycles of overlap extension PCR were taken for agarose gel electrophoresis. The optimal PCR cycle or optimal combination of different PCR cycles was chosen according to the results of electrophoresis. Then 100 bp DNA ladder marker was obtained by the overlap extension PCR under the chosen condition. There was no need for purification of PCR products as the amplified fragments could be directly used in the agarose gel electrophoresis. The bands of prepared DNA marker were clear and accurate, and could be used for molecular studies. In sum, this method for DNA marker production is simple, time saving, cost effective, byproduct free and user-customizable in comparison with current ones widely used in most laboratories.
引文
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[11]李梅,廖龙,林惠超,等.基于PCR技术的DNA marker的研制.中国生物制品学杂志,2017,30(7):769-771.LI M,LIAO L,LIN H C,et al.Preparation of DNA marker by PCR technology.Chinese Journal of Biologicals,2017,30(7):769-771.(in Chinese)
[12]张俊河,王俐,董卫华,等.基于多重PCR的DNA marker制备方法.生物技术通报,2010,26(12):189-190,200.ZHANG J H,WANG L,DONG W H,et al.A simple method for preparing DNA marker.Biotechnology Bulletin,2010,26(12):189-190,200.(in Chinese with English abstract)
[13]CHANG M,WANG J H,LEE H J.Laboratory production of100 base pair DNA molecular weight markers.Journal of Biochemical and Biophysical Methods,2008,70(6):1199-1202.
[14]HUANG D Y,ZHOU L H,ZENG H Z,et al.Construction of DNA marker plasmids based on Taq tailing activity and selective recovery of ligation products.Plant Molecular Biology Reporter,2008,26:316-323.
[15]LAN V T T,LOAN P T T,DUONG P A T,et al.Straightforward procedure for laboratory production of DNAladder.Journal of Nucleic Acids,2012(3):254630.
[16]刘慧娟,冯志国,何光源.利用PCR技术快速制备DNA分子量标准物.生物技术,2008,18(2):38-39.LIU H J,FENG Z G,HE G Y.A method for fast preparation of DNA marker by PCR technology.Biotechnology,2008,18(2):38-39.(in Chinese with English abstract)
[17]吕金浮,乔宁,刘永光,等.基于PCR技术快速制备DNAmarker的研究.河南农业科学,2014,43(8):149-151,156.LüJ F,QIAO N,LIU Y G,et al.Study on rapid preparation of DNA marker by PCR technology.Journal of Henan Agricultural Sciences,2014,43(8):149-151,156.(in Chinese with English abstract)
[18]王文华,葛常辉.一种简便的DNA定量分子量标准的制备.生物技术,2010,20(3):62-64.WANG W H,GE C H.A simple preparation of DNA mass MW marker.Biotechnology,2010,20(3):62-64.(in Chinese with English abstract)
[19]韦柳静,韦宇拓,黄小凤,等.一种新型可用于DNA分子量标准的质粒构建.生物技术,2004,14(5):33-35.WEI L J,WEI Y T,HUANG X F,et al.Construction of a new kind of DNA marker vector.Biotechnology,2004,14(5):33-35.(in Chinese with English abstract)
[2]张颖,刘发珍,李强,等.DNA分子量标准制备技术:方法与进展.中国生物工程杂志,2009,29(8):119-123.ZHANG Y,LIU F Z,LI Q,et al.Preparation of DNA molecular weight standards:methods and advances.China Biotechnology,2009,29(8):119-123.(in Chinese with English abstract)
[3]ZHANG J H,YANG R,WANG T Y,et al.A simple and practical method that prepares high molecular weight DNAladders.Molecular Medicine Reports,2012,6(5):1211-1213.
[4]韩荣荣,吴永红,屈武斌,等.利用单线虫多重PCR技术快速制备DNA分子量标准.西北农业学报,2012,21(3):12-16.HAN R R,WU Y H,QU W B,et al.A method for rapid preparation of DNA molecular weight standard by single C.elegans multiplex PCR.Acta Agriculturae Boreali-Occidentalis Sinica,2012,21(3):12-16.(in Chinese with English abstract)
[5]CHEN Z,WU J B,LI X J,et al.Novel strategies to construct complex synthetic vectors to produce DNAmolecular weight standards.Molecular Biotechnology,2009,42(1):128-133.
[6]MOSTAAN S,AJORLOO M,KHANAHMAD H,et al.Anovel combined method for cost-benefit production of DNAladders.Advanced Biomedical Research,2015,4:15-18.
[7]SEKHAVATI M,TADAYON K,GHADERI R,et al.“Inhouse”production of DNA size marker from a vaccinal Bacillus anthracis strain.Iranian Journal of Microbiology,2015,7(1):45-49.
[8]ABBASIAN M,SEYEDI H A E,BOROUJENI Z K,et al.Easy method for production of a home-made DNA ladder in every laboratory.Advanced Biomedical Research,2015,4:70-74.
[9]王俐,董卫华,张俊河,等.DNA ladder的制备.中国组织工程研究与临床康复,2011,15(24):4493-4496.WANG L,DONG W H,ZHANG J H,et al.Preparation of DNA ladder.Journal of Clinical Rehabilitative Tissue Engineering Research,2011,15(24):4493-4496.(in Chinese with English abstract)
[10]李文成,杨博,王永华.一种用于形成1 kb阶梯DNA分子量标准载体的构建.湖北农业科学,2009,48(1):1-3.LI W C,YANG B,WANG Y H.Construction of vectors for creating 1 kb ladder DNA molecular weight standards.Hubei Agricultural Sciences,2009,48(1):1-3.(in Chinese with English abstract)
[11]李梅,廖龙,林惠超,等.基于PCR技术的DNA marker的研制.中国生物制品学杂志,2017,30(7):769-771.LI M,LIAO L,LIN H C,et al.Preparation of DNA marker by PCR technology.Chinese Journal of Biologicals,2017,30(7):769-771.(in Chinese)
[12]张俊河,王俐,董卫华,等.基于多重PCR的DNA marker制备方法.生物技术通报,2010,26(12):189-190,200.ZHANG J H,WANG L,DONG W H,et al.A simple method for preparing DNA marker.Biotechnology Bulletin,2010,26(12):189-190,200.(in Chinese with English abstract)
[13]CHANG M,WANG J H,LEE H J.Laboratory production of100 base pair DNA molecular weight markers.Journal of Biochemical and Biophysical Methods,2008,70(6):1199-1202.
[14]HUANG D Y,ZHOU L H,ZENG H Z,et al.Construction of DNA marker plasmids based on Taq tailing activity and selective recovery of ligation products.Plant Molecular Biology Reporter,2008,26:316-323.
[15]LAN V T T,LOAN P T T,DUONG P A T,et al.Straightforward procedure for laboratory production of DNAladder.Journal of Nucleic Acids,2012(3):254630.
[16]刘慧娟,冯志国,何光源.利用PCR技术快速制备DNA分子量标准物.生物技术,2008,18(2):38-39.LIU H J,FENG Z G,HE G Y.A method for fast preparation of DNA marker by PCR technology.Biotechnology,2008,18(2):38-39.(in Chinese with English abstract)
[17]吕金浮,乔宁,刘永光,等.基于PCR技术快速制备DNAmarker的研究.河南农业科学,2014,43(8):149-151,156.LüJ F,QIAO N,LIU Y G,et al.Study on rapid preparation of DNA marker by PCR technology.Journal of Henan Agricultural Sciences,2014,43(8):149-151,156.(in Chinese with English abstract)
[18]王文华,葛常辉.一种简便的DNA定量分子量标准的制备.生物技术,2010,20(3):62-64.WANG W H,GE C H.A simple preparation of DNA mass MW marker.Biotechnology,2010,20(3):62-64.(in Chinese with English abstract)
[19]韦柳静,韦宇拓,黄小凤,等.一种新型可用于DNA分子量标准的质粒构建.生物技术,2004,14(5):33-35.WEI L J,WEI Y T,HUANG X F,et al.Construction of a new kind of DNA marker vector.Biotechnology,2004,14(5):33-35.(in Chinese with English abstract)