Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Construction and Analysis of Genomic Libraries
Construction of Genomic Libraries and Gene Identification Therein

In most cases, studying the Structure and properties of a specific Gene in an Organism requires a preliminary step: constructing a gene bank for that organism. A gene bank, or genomic library (often called a clone library), is a collection of bacterial clones or phage particles containing at least one copy of each sequence in the organism's genome. The required number of clones in a library is determined by The ratio of the Genome Size to the size of the cloned DNA fragments. For example, if the haploid genome of Saccharomyces cerevisiae contains 1.4∙10⁷ bp, and the capacity of the cloning vector used is 15∙10³ bp, the entire Yeast genome can be represented by 1.4∙10⁷ /15∙10³ ≈ 1∙10³ recombinant clones. In practice, however, creating a reliable library in this case requires 4,000–5,000 clones to ensure a 99% probability of finding the target gene or genome segment. Such redundancy is necessary because the ligation of individual foreign DNA fragments with vectors occurs randomly; consequently, some genomic regions may be represented multiple times in a small sample, while others may not appear at all. A complete human genomic library, for instance, consists of approximately 900,000 clones.

The two most frequently used types of clone libraries are Genomic Libraries and cDNA libraries. Genomic libraries are collections of all the METABOLISM/27.html">Genes and DNA sequences of an organism. They are typically generated using vectors constructed from bacteriophage λ or cosmids. These vectors are characterized by a large cloning capacity, which helps minimize the total number of clones required in the library.

A cDNA library (“complementary” DNA, see Section 1) consists of a set of clones containing double-stranded DNA copies of all cellular mRNAs. Plasmid or phage-based vectors (derived from phage λ) are most commonly used to construct these libraries.

Both types of libraries, however, can be likened to a chaotic collection of books that only becomes a true library once a catalog is compiled to systematically organize the entire collection. In other words, the next crucial step is the identification of genes within the genomic library. This can be accomplished in several ways: by analyzing the inserted polynucleotide sequence itself or by examining its expression products in host Cells. In the first approach, the screening strategy for recombinant clones relies on probes complementary to specific Regions of the genes or cDNA. In the second approach, screening is based either on Changes in the phenotype of cells that have taken up a specific DNA fragment—driven by the newly synthesized protein—or simply on The properties of the protein itself. Let us examine some of the Methods used to identify genes within clone libraries.

Gene identification via altered cellular phenotype. This method is most commonly used in “self-cloning,” i.e., when genes are transferred via multi-copy Plasmids back into the same microorganism from whose genome they were originally isolated. For example, suppose one needs to find E. coli clones in a library that contain the genes responsible for Alanine Biosynthesis. This requires transferring vectors carrying cloned DNA fragments into mutant E. coli strains that are alanine-dependent. Progeny Selection is then performed on a synthetic medium lacking alanine, where only those Bacteria that have taken up the gene complementing the corresponding mutation are able to form colonies. This Procedure is known as a “complementation test” and requires strains with well-characterized Mutations in the target genes.

Immunological screening of gene products. If DNA fragments were cloned within expression vectors that ensure the Transcription and Translation of foreign genes, the desired clones can be identified using immunological tests. To do this, bacterial colonies grown on solid medium (a replica plate) are lysed using chloroform vapors, and their contents are transferred by replica plating onto a polyvinyl sheet coated with Antibodies against the target protein. The sheet is then washed under mild conditions so that only the Proteins bound to the antibodies remain. Next, the membrane is treated with radioactively labeled (¹²⁵I) antibodies specific to the same protein. As a result, a complex forms on the membrane consisting of the antigen (the target protein) and two antibody molecules, one of which is radiolabeled. The Location of this complex can be detected by autoradiography. By correlating THE POSITION OF the labeled complex with the positions of the colonies on the master plate, researchers can identify the clone containing the gene responsible for synthesizing the target protein.

Screening a clone library using probes. Section 1 of this chapter describes the procedure for obtaining cDNA. To search for a desired cDNA within a library, the following approach is used. An isolated colony containing the vector with the insert is selected and incubated to produce a large, homogeneous culture. Plasmid DNA is then extracted from the cells and denatured to separate its strands. The denatured DNA is immobilized on a nitrocellulose filter. A mixture of mRNAs, extracted from the same cells whose genes are being sought, is passed through the filter. Under these conditions, only those mRNAs that are complementary to the homogeneous cDNA are retained on the filter. The bound mRNA is eluted from the filter and introduced into a Cell-free translation system, where the corresponding protein is synthesized. This protein can then be identified immunologically or via chromatographic analysis. Once successful, the researcher has an identified clone whose cells contain the cDNA, which can subsequently serve as a probe to search for the individual gene or mRNA.

A cDNA probe can be used for rapid and highly efficient screening of any clone library. The experimental setup for this procedure is illustrated in Fig. 20.8. Isolated microorganism colonies or virus plaques growing on a lawn of sensitive cells are transferred via replica plating onto a nitrocellulose filter placed On the surface of solid medium in a Petri dish. The cells are lysed (typically via alkaline lysis using NaOH) and their DNA is denatured. Under specific conditions (80°C, vacuum), the DNA binds firmly to the nitrocellulose filter. The filter is then incubated in a solution containing a denatured ³²P-labeled probe. Under these conditions, complementary DNA sequences form duplexes. Autoradiographic analysis reveals darkening at the exact spot on the filter where the DNA complementary to the probe is located. Researchers then match this zone back to the corresponding colony (or plaque) on the master plate.

Instead of cDNA, mRNA or chemically synthesized oligonucleotide probes can also be used to screen clone libraries. The latter approach requires prior knowledge of The nucleotide sequence of the gene or the Amino Acid Sequence of the target protein. Based on The amino acid sequence of a polypeptide segment 5–6 residues long, all possible mRNA sequences capable of encoding that segment are predicted. Because of the degeneracy of The Genetic Code, there are usually many such variant sequences. A corresponding set of complementary oligonucleotides is then synthesized in vitro. These NUCLEOTIDES can be used directly to screen a cDNA mixture or a genomic library. Alternatively, these oligonucleotides can serve as primers—replacing oligothymidylates—for Reverse Transcriptase to synthesize cDNA substantially enriched in the target sequences. In this case, the reverse transcription process will predominantly copy those mRNA molecules that contain sequences complementary to the primers.

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Fig. 20.8. Schematic diagram of clone library screening using probes