Cloning of Enhancer of Zeste Homolog 2 in Forward Orientation Into Escherichia Coli Using Histidine-Tagged Pbluescript Ii Ks+.
Title: Cloning of Enhancer of Zeste Homolog 2 in forward orientation into Escherichia Coli using histidine-tagged pbluescript II KS+.
Abstract
Enhancer of Zeste Homolog 2 locus is intensely over expressed in breast and prostate cancer and it’s been established that its promoter inhibition by p53 has led to reduced cell proliferation and invasion (Bracken, 2003; Xiao, 2011). Objective is to clone a forward orientated EZH2 insert into a his-tagged pbluescript. Cloning EZH2 into a histidine-tagged pbluescript in a forward orientation potentially allows isolation of protein via Affinity Chromatography or Chromatin Immunoprecipitation therefore its role, effects and targets in the genome can be established. Resultant Recombinant plasmids in this experiment had multiple inserts leading to inconclusive orientation of the inserts; however this can be tackled by Sanger or Maxam/Gilberts sequencing.
Introduction
The capacity to segregate and amplify individual genes from an intricate genome using recombinant DNA technology technique like PCR has profoundly influenced ways scientists explored the obscurities in biology. Some of the major accomplishments of Recombinant DNA technology include the synthesis of medically essential proteins like insulin and developing new vaccines routes (Kingsman and Kingsman, 1988; Primrose et al., 2001).
Gene Cloning allows the amplification of target sequences (e.g. Enhancer of Zeste Homolog 2/ insert) transferred as part of a vector (his- tagged pbluescript) into Escherichia Coli which reproduces asexually yielding progenies that contain the desired recombinant plasmid (Dale and Schantz, 2007). pbluescript is a bacterial based relaxed phagemid (~2.9kb), derived from pUC 19 which has 2 origins of replication (f1 and Col E1), 2 selectable marker Lac Z´ and ampR gene and due to its mutated RNA I it has a high copy number. It’s competent as cloning vector because it contains a multiple cloning site and transcribed by bacterial RNA
Abstract
Enhancer of Zeste Homolog 2 locus is intensely over expressed in breast and prostate cancer and it’s been established that its promoter inhibition by p53 has led to reduced cell proliferation and invasion (Bracken, 2003; Xiao, 2011). Objective is to clone a forward orientated EZH2 insert into a his-tagged pbluescript. Cloning EZH2 into a histidine-tagged pbluescript in a forward orientation potentially allows isolation of protein via Affinity Chromatography or Chromatin Immunoprecipitation therefore its role, effects and targets in the genome can be established. Resultant Recombinant plasmids in this experiment had multiple inserts leading to inconclusive orientation of the inserts; however this can be tackled by Sanger or Maxam/Gilberts sequencing.
Introduction
The capacity to segregate and amplify individual genes from an intricate genome using recombinant DNA technology technique like PCR has profoundly influenced ways scientists explored the obscurities in biology. Some of the major accomplishments of Recombinant DNA technology include the synthesis of medically essential proteins like insulin and developing new vaccines routes (Kingsman and Kingsman, 1988; Primrose et al., 2001).
Gene Cloning allows the amplification of target sequences (e.g. Enhancer of Zeste Homolog 2/ insert) transferred as part of a vector (his- tagged pbluescript) into Escherichia Coli which reproduces asexually yielding progenies that contain the desired recombinant plasmid (Dale and Schantz, 2007). pbluescript is a bacterial based relaxed phagemid (~2.9kb), derived from pUC 19 which has 2 origins of replication (f1 and Col E1), 2 selectable marker Lac Z´ and ampR gene and due to its mutated RNA I it has a high copy number. It’s competent as cloning vector because it contains a multiple cloning site and transcribed by bacterial RNA
References: Alting-Mees .M.A, Short .J.M, 1989, Pbluescript II: gene mapping vectors, Nucleic Acid Research, 17(22), available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC335171/pdf/nar00139-0575.pdf Accessed on 1st December, 2011. Primrose .S.B, Twyman .R.M, Old .R.W, 2001, Principles of Gene Manipulation, Oxford: Blackwell Science Ltd Promega, 2011, EcoR1, Available at: http://www.promega.com/products/cloning-and-dna-markers/restriction-enzymes/ecori/ Accessed 26th Nov 2011 Qiagen, 2011, the Purification Kit, Available at: http://www.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRP urificationKit.aspx?r=1745#Tabs=t2, Accessed 26th Nov 2011 Sambrook .J, Russell .W.D, 2001, Molecular Cloning: A laboratory Manual, 3rd Ed, Vol2, USA: Cold Spring Harbour Laboratory Press UniProtkb, 2011, EZH2 –Human, Available at: http://www.uniprot.org/uniprot/Q15910 Accessed 26th Nov 2011 Vennison .J.S, 2009, Laboratory Manual for Genetical Engineers, New Delhi: Phi learning private Ltd Walker .R.M, Rapley .R, 1997, Route maps in Gene technology, UK: Blackwell Science Ltd