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未知侧翼序列扩增-DNA Walking步移法

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DNA WALKING REFERRENCEKi-Bum Park and Suk-Heung Oh(2007)Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3. Bioresource Technology 98(2): 312-319.

Young-Jun Park, Soo Young Choi, and Hee-Bong Lee(2006)A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression.Biochimica et Biophysica (BBA) - General Subjects 1760(5):820-828.

Andrey A. Perelygin, Andrey A. Zharkikh, Svetlana V. Scherbik and Margo A. Brinton (2006)The Mammalian 2′-5′ Oligoadenylate Synthetase Gene Family: Evidence for Concerted Evolution of Paralogous Oas1 Genes in Rodentia and Artiodactyla. Journal of Molecular Evolution 63(4):562-576.

C. Nisbet, I. Butzonitch, M. Colavita, J. Daniels, J. Martin, R. Burns, E. George, M. A. Y. Akhond, V. Mulholland, and C. J. Jeffries (2006)Characterization of Potato rough dwarf virus and Potato virus P: distinct strains of the same viral species in the genus Carlavirus.Plant Pathology 55(6): 803.

Koo BC, Kwon MS, Choi BR, Kim JH, Cho SK, Sohn SH, Cho EJ, Lee HT, Chang W, Jeon I, Park JK, Park JB, Kim T.(2006)Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector.FASEB J. 20(13):2251-60.

Barbet AF, Lundgren AM, Alleman AR, Stuen S, Bjoersdorff A, Brown RN, Drazenovich NL, Foley JE.(2006)Structure of the expression site reveals global diversity in MSP2 (P44) variants in Anaplasma phagocytophilum. Infect. Immun. 74(11):6429-37.

Chunhong Yan and Douglas D. Boyd(2006)Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression. Molecular and Cellular Biology 26(17):6357-6371.

Alhapel A, Darley DJ, Wagener N, Eckel E, Elsner N, Pierik AJ.(2006)Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri.Proc Natl Acad Sci U.S.A. 103(33):12341-6.

Knopf RR and Trebitsh T.(2006)The female-specific Cs-ACS1G gene of cucumber. A case of gene duplication and recombination between the non-sex-specific 1-aminocyclopropane-1-carboxylate synthase gene and a branched-chain amino acid transaminase gene. Plant Cell Physiol. 47(9):1217-28..

Qizhen Shi, David A. Wilcox, Scot A. Fahs, Hartmut Weiler, Clive W. Wells, Brian C, Cooley, Drashti Desai, Patricia A. Morateck, Jack Gorski, and Robert R. Montgomery (2006)Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies. The Journal of Clinicel Investigation 116( 7):1974-1982.

Qizhen Shi, David A. Wilcox, Scot A. Fahs, Hartmut Weiler, Clive W. Wells, Brian C. Cooley, Drashti Desai, Patricia A. Morateck, Jack Gorski, and Robert R. Montgomery (2006)Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies. J. Clin. Invest. 116(7):1974-1982.

Ahu Altinkut, Olga Raskina, Eviatar Nevo and Alexander Belyayev(2006)

En/Spm-like transposons in Poaceae species: Transposase sequence variability and chromosomal distribution. Cellular & Molecular Biology Letters Issue 11(2)214-229.

Assaf Distelfield, Cristobal Uauy, Tzion Fahima, and Jorge Dubcovsky (2006)

Physical map of the wheat high-grain protein content gene Gpc-B1 and development of a high-throughput molecular marker. New Phytologist 169:753-763.

Andrey A. Perelygin, Teri L. Lear, Andrey A. Zharkikh and Margo A. Brinton (2006)

Comparative analysis of vertebrate EIF2AK2 (PKR) genes and assignment of the equine gene to ECA15q24-q25 and the bovine gene to BTA11q12-q15. Genet. Sel. Evol. 38:551-563.

Jung-Wook Kim, Yasuo Yamakoshi, Takanori Iwata, Yuan Yuan Hu, Hengmin Zhang, Jan C.-C. Hu, James P. Simmer (2006)

Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth. European Journal Of Oral Sciences114(1):33-41.

Chengshu Wang and Raymond J. St. Leger(2006)

A collagenous protective coat enables Metarhiziumanisopliae to evade insect immune responses. PNAS 103:6647-6652.

EMIKO HAYAMA, SHIN-ICHIRO IMAMURA, CUIJIAO WU, MAKOTO NAKAZAWA, RUMIKO MATSUOKA and TOSHIO NAKANISHI(2006)

Analysis of Voltage-Gated Potassium Channel ß1 Subunits in the Porcine Neonatal Ductus Arteriosus. Pediatric Research 59:167-174.

M.-C. Domingo, A. Huletsky, A. Bernal, R. Giroux, D. K. Boudreau, F. J. Picard and M. G. Bergeron(2005)

Characterization of a Tn5382-like transposon containing the vanB2 gene cluster in a Clostridium strain isolated from human faeces. Journal of Antimicrobial Chemotherapy 55(4):466-474.

Perelygin AA, Lear TL, Zharkikh AA, Brinton MA (2005)

Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15 p14 and BTA17q24q25. Cytogenet Genome Res 111(1):51-6.

Park, M., H. J. Shin, S. Y. Lee, and T. I. Ahn.(2005)

Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus. J Eukaryot Microbiol 52:223-30.

科研人员已经发展了几种无需建立cDNA 库或筛选克隆的PCR 技术方法,用于获取与已知序列相连的未知序列。这些PCR 方法包括反向PCR (IPCR ),连接介导PCR (LM-PCR )和随机引物PCR (RP-PCR )。虽然这些方法有一定的效果并被不断改进,但是仍然受制于繁琐的酶切,适配体连接 (adaptor ligation)和纯化步骤。而且,RP-PCR 的随机引物经常会因为非特异性退火而产生多余产物。DNA 步移法加速试剂盒通过应用专利的ACP(复性控制引物)技术,提高了小片断寡聚核苷酸的特异性,可以克服现有基因步移法的缺点。

DNA 步移法加速试剂盒由PCR 酶混合物与用于捕获未知目标序列的高特异DNA Walking ACP (DW-ACP)引物组成。据此优化的PCR 反应条件(以下称为DW ACP-PCR 技术)可以获取长达3kb的未知侧翼序列。

一种全新的扩增未知目标序列的快速PCR 方法。

试剂盒由PCR Master Mix和专为高特异度捕捉未知靶点的独特DNA 步移退火控制引物(DW-ACPs)组成。此最优化的PCR 条件,称为 DW ACP-PCR TM技术,可以获取长达3kb的未知侧区。此试剂盒是直接扩增未知序列的革命性方法。

"Nature 'Product focus-Transgenics' September 7, 2006"!

特点:

*最简单

此方法无须建库,无须酶切,无须固定,也无须复杂PCR

*最快捷

你的目标产物可在一天内获得,此方法超越其他现有方法

*最可靠

只获取目标产物

应用范围:

● 基因组步移

- 启动子区域的克隆或者测序

- 基因结构(外显子/内含子结合区)

- 已知序列上下游侧翼的扩增

- cDNA 步移

- 裂隙填补(Gap Filling)

- 序列标签位点(STSs)和表达序列标记(EST)的双向测序

● 转基因位点检测

- 在转基因生物如转基因植物,动物,昆虫,鱼和细菌中发现转基因或T-DNA 的位置或导向

- 直接扩增并分离出具有转基因或T-DNA 的侧翼区域的DNA 片断

● 缺失/插入/突变体的检测

- 检测基因组DNA 或cDNA 的缺失,插入,或者突变体

- 剪切分析

● 测序

- BAC / PAC DNA 的末端测序或者步移

- BACs或者基因组DNA 的直接测序

- 基因组DNA ,cDNA 或质粒DNA (BAC, PAC, YAC)的测序

- 无须亚克隆或者鸟枪克隆即可对BAC或PAC克隆测序

- 长链DNA 的快速测序

- 基因组测序计划*基因组步移

-启动子区域的克隆或者测序

-基因结构(外显子/内含子结合区)

-裂隙填补(Gap Filling)

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