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3: Isolating and Analyzing Genes

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  • The first two chapters covered many important aspects of genes, such as how they function in inheritance, how they code for protein (in general terms) and their chemical nature. All this was learned without having a single gene purified. A full understanding of a gene, or the entire set of genes in a genome, requires that they be isolated and then studied intensively. Once a gene is “in hand”, in principal one can determine both its biochemical structures and its function(s) in an organism. One of the goals of biochemistry and molecular genetics is to assign particular functions to individual or composite structures. This chapter covers some of the techniques commonly used to isolate genes and illustrates some of the analyses that can be done on isolated genes.

    • 3.1: Recombinant DNA, Polymerase Chain Reaction and Applications to Eukaryotic Gene Structure and Function
    • 3.2: Overview of Recombinant DNA Technology
    • 3.3: Introduction of recombinant DNA into cell and replication: Vectors
      Vectors used to move DNA between species, or from the lab bench into a living cell, must meet three requirements:  (1) They must be autonomously replicating DNA molecules in the host cell.  (2) They must contain a selectable marker so cells containing the recombinant DNA can be distinguished from those that do not. (3) They must have an insertion site to accommodate foreign DNA. Usually a unique restriction cleavage site in a nonessential region of the vector DNA.
    • 3.4: Introducting Recombinant DNA into Host Cells
    • 3.5: Polymerase Chain Reaction (PCR)
      The polymerase chain reaction (PCR) is now one of the most commonly used assays for obtaining a particular segment of DNA or RNA. It is rapid and extremely sensitive. By amplifying a designated segment of DNA, it provides a means to isolate that particular DNA segment or gene. This method requires knowledge of the nucleotide sequence at the ends of the region that you wish to amplify.
    • 3.6: cDNA
      Construction of cDNA clones involves the synthesis of complementary DNA from mRNA and then inserting a duplex copy of that into a cloning vector, followed by transformation of bacteria.
    • 3.7: Genomic DNA clones
      Clones of genomic DNA, containing individual fragments of chromosomal DNA, are needed for many purposes
    • 3.8: Eukaryotic Gene Structure
      Much can be learned about any gene after it has been isolated by recombinant DNA techniques. The structure of coding and noncoding regions, the DNA sequence, and more can be deduced. This is true for bacterial and viral genes, as well as eukaryotic cellular genes. The next sections of this chapter will focus on analysis of eukaryotic genes, showing the power of examining purified copies of genes.
    • 3.9: Introns and Exons
      Far more exons and introns have been discovered (or more accurately, predicted) throught the analysis of genomic DNA sequences than could ever be discovered by direct experimentation. The different types of exons, the enormous length of introns, and other factors have complicated the task of finding reliable diagnostic signatures for exons in genomic sequences. However, considerable progress has been made and continues in current research.
    • 3.10: Functional analysis of isolated genes
    • 3.E: Isolating and Analyzing Genes (Exercises)

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