Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard Microalgal pcna as the Model Gene 英文参考文献.docVIP

下载本文档

13
0
约3.31万字
约 7页
2017-05-13 发布于上海
举报
版权申诉

Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard Microalgal pcna as the Model Gene 英文参考文献.doc

1、本文档共7页，可阅读全部内容。
2、原创力文档（book118）网站文档一经付费（服务费），不意味着购买了该文档的版权，仅供个人/单位学习、研究之用，不得用于商业用途，未经授权，严禁复制、发行、汇编、翻译或者网络传播等，侵权必究。
3、本站所有内容均由合作方或网友上传，本站不对文档的完整性、权威性及其观点立场正确性做任何保证或承诺！文档内容仅供研究参考，付费前请自行鉴别。如您付费，意味着您自己接受本站规则且自行承担风险，本站不退款、不进行额外附加服务；查看《如何避免下载的几个坑》。如果您已付费下载过本站文档，您可以点击这里二次下载。
4、如文档侵犯商业秘密、侵犯著作权、侵犯人身权等，请点击“版权申诉”（推荐），也可以打举报电话：400-050-0827(电话支持时间：9:00-18:30)。
5、该文档为VIP文档，如果想要下载，成为VIP会员后，下载免费。
6、成为VIP后，下载本文档将扣除1次下载权益。下载后，不支持退款、换文档。如有疑问请联系我们。
7、成为VIP后，您将拥有八大权益，权益包括：VIP文档下载权益、阅读免打扰、文档格式转换、高级专利检索、专属身份标志、高级客服、多端互通、版权登记。
8、VIP文档为合作方或网友上传，每下载1次，网站将根据用户上传文档的质量评分、类型等，对文档贡献者给予高额补贴、流量扶持。如果你也想贡献VIP文档。上传文档

Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard Microalgal pcna as the Model Gene 英文参考文献

SeriousOverestimationinQuantitativePCRbyCircular (Supercoiled)PlasmidStandard:Microalgalpcnaasthe ModelGene YuboHou¤a*,HuanZhang,LilibethMiranda¤b,SenjieLin DepartmentofMarineSciences,UniversityofConnecticut,Groton,Connecticut,UnitedStatesofAmerica Abstract Quantitativereal-timePCR(qPCR)hasbecomeagoldstandardforthequantificationofnucleicacidsandmicroorganism abundances, in which plasmidDNA carrying the targetgenes aremost commonly used as the standard. A recentstudy showedthatsupercoiledcircularconfirmationofDNAappearedtosuppressPCRamplification.However,towhatextentto whichdifferentstructuraltypesofDNA(circularversuslinear)usedasthestandardmayaffectthequantificationaccuracy hasnotbeenevaluated.Inthisstudy,wequantitativelycomparedqPCR accuraciesbased oncircular plasmid(mostlyin supercoiledform)andlinearDNAstandards(linearizedplasmidDNAorPCRamplicons),usingproliferatingcellnucleargene (pcna),theubiquitouseukaryoticgene,infivemarinemicroalgaeasamodelgene.WeobservedthatPCRusingcircular plasmidsastemplategave2.65-4.38moreofthethresholdcyclenumberthandidequimolarlinearstandards.Whilethe documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circularplasmidasstandardyieldedanestimateof7.77copiesofpcnapergenomewhereasthatusingthelinearstandard gave1.02copiespergenome.WeconcludethatcircularplasmidDNAisunsuitableasastandard,andlinearDNAshouldbe usedinstead,inabsoluteqPCR.Theseriousoverestimationbythecircularplasmidstandardislikelyduetotheundetected lowerefficiencyofitsamplificationintheearlystageofPCRwhenthesupercoiledplasmidisthedominanttemplate. Citation:HouY,ZhangH,MirandaL,LinS(2010)SeriousOverestimationinQuantitativePCRbyCircular(Supercoiled)PlasmidStandard:Microalgalpcnaasthe ModelGene.PLoSONE5(3):e9545.doi:10.1371/journal.pone.0009545 Editor:SharynJaneGoldstien,UniversityofCanterbury,NewZealand ReceivedNovember14,2009;AcceptedFebruary14,2010;PublishedMarch5,2010 Copyright:?2010Houetal.Thisisanopen-accessart