Cloning in Plasmid Vectors, palindromic Sequences, Previous Moleculer Similarly Treated, Chimeric DNA, Chimeric Molecules, Plasmid Vector

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Chimeric DNA Molecular Probes and Gene Libraries Chimeric DNA Molecular Probes and Gene Libraries Introduction Restriction Enzymes for Cloning Technique of Restriction Mapping Restriction Clevage and Gel Electrophoresis Construction of a Restriction Map Use of Partial Digests end Labelling and Hybridization in Restriction Mapping Construction of Chimeric DNA Cloning of Plasmid Vectors Directional Cloning of DNA Fragments with Heterologus Protruding Termini Adding Poly dA at 3' Ends of DNA Clone Blunt End Ligation by T4 DNA Ligase Cloning in Phage λ and Cosmid Vectors Cloning in phage λ Vectors Cloning in Cosmid Vectors Hosts for Cloning Vectors Bacteria as Hosts Eukaryotes as Hosts Molecular Probes Preparation of Probes Genomic DNA Probes

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Your Ad Here	Cloning in plasmid vectors Cloning DNA fragments with homologous cohesive ends. This is the most common method, where due to the presence of a palindromic sequence, a restriction enzyme causes staggered cuts producing short complementary sequences at the two ends of (a palindromic sequence is one, which has complementary sequences at the two ends of a single strand, e.g. ATC GAT).
This can be illustrated using the example of enzyme EcoRI, which cuts the sequences at specific positions. When another DNA molecule is similarly cut by the same enzyme, similar sticky ends having same sequences in the single stranded ends will be produced, so that when it is mixed with the previous molecule similarly treated, the two will anneal producing a chimeric DNA or chimeric plasmid, if plasmid DNA is involved Staggered cleavage of a double stranded DNA fragement, producing single stranded sticky ends
Enzyme DNA ligase helps in joining the bonds at the cut ends of the two molecules. This method of cloning is commonly employed with pBR322 and pUC vectors. The above technique has the advantage of regenerating two restriction sites (e.g. EcoRI sites) in the chimeric DNA, so that the foreign DNA segment can be retrieved rather easily from the cloned copies of chimeric DNA by cleavage again with the same enzyme. There are also following disadvantages with this technique. (i) The cleaved ends of two vector molecules or those of two foreign DNA segments may join end to end before the foreign DNA gets inserted.	Your Ad Here
Therefore, while isolating chimeric DNA, one will have to select chimeric molecules each having only a single insert. This can be achieved by separating molecules of different sizes by gel electrophoresis. Cloning of a DNA fragment in a plasmid vector by Stagged cleavages, both in Vector DNA and foreign DNA to be Cloned This difficulty can be overcome, firstly, by adjusting the concentrations of two types of DNA and secondly, by removing 5' phosphate groups in the linearized vector. (ii) The recognition site, particularly in the sequence to be cloned may not lie at a convenient position. so that sometimes only a part of the desired segment will be inserted.

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c DNA Probes Synthetic Oligonucleotides as Probes RNA Probes or Riboprobes Labelling of Probes Radiolablled Probes Non Radioactive Probes Biotin Labelled Probes Digoxigenin Labelled Probes Alternatives to Biotin and Digoxigenin Labelling Amplification of DNA Probe Signals Techniques Used in Molecular Probing Southern Nothern and Western Blotting Southern Blotting Northern Blotting Western Blotting Dot Blots and Slot Blots Applications of Molecular Probes Construction and Screening of Genomic and cDNA Libraries Genomic Library by Shotgun Experiment cDNA Library from mRNAs Colony or Plaque Hybridization for Screening of Libraries Chromosome Walking and Characterization of Chromosome Segments Chromosome Jumping or Hopping Libraries BAC Libraries and Assembly of BACs into Contigs