Genetics - A. V. Sivolob 2008

Genetic Engineering and Methods of Molecular Genetics
Analysis of Gene and Genome Structure and Expression

A cloned or amplified DNA fragment can be studied in various ways, but the most comprehensive information is provided by determining its nucleotide sequence—a process known as sequencing.

Figure 9.6 illustrates the layout of the Sanger method (developed by Frederick Sanger), which remains the most popular today. A radioactively labeled primer, a full set of deoxynucleoside triphosphates (dNTPs), DNA polymerase, and a small amount of a dideoxynucleoside triphosphate of a single type (e.g., ddATP) are added to a single-stranded DNA template. Dideoxynucleotides differ by containing an H atom instead of an OH group at both the 2' and 3' positions of the pentose ring (see Fig. 1.1). Consequently, the incorporation of such a nucleotide into the synthesized chain halts further elongation due to the lack of a 3' OH group at its terminus. Because ddATP is present in a limited amount, this termination event occurs randomly at all positions where adenine is incorporated opposite a thymine in the template strand. Denaturation of the reaction products yields a set of labeled single-stranded fragments extending from the primer to the terminal adenine; the length of these fragments in NUCLEOTIDES directly reveals the exact position of the adenine within the chain.

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Fig. 9.6. Sanger DNA Sequencing: scheme of DNA Synthesis in the presence of dideoxyATP (left). An analogous Procedure using the other three dideoxynucleotides generates a set of single-stranded fragments analyzed by denaturing gel Electrophoresis (right)—band Separation allows the sequence to be read (bottom right)

To determine fragment lengths, denaturing gel electrophoresis of the single-stranded DNA is performed, with the synthesis products of the other dideoxynucleotides loaded into adjacent gel lanes. As shown in Fig. 9.6, The nucleotide sequence can be read directly from the gel following electrophoresis and band visualization.

Another modern sequencing approach, known as pyrosequencing, is carried out on automated sequencers and allows sequences to be determined much faster and more affordably, without requiring either DNA Cloning or electrophoresis. Single-stranded fragments obtained from a small amount of genomic DNA are attached via their 5' ends to microbeads (one fragment per bead) and amplified by PCR. Each bead, covered with amplified identical fragments, is placed into a microreactor where the DNA polymerase reaction takes place. Nucleoside triphosphates are pulsed into the reaction mixture one by one. If a nucleotide of a specific type is complementary to the template and incorporated into the growing strand, the released pyrophosphate drives a cascade of Chemical Reactions, the final step of which emits light (chemiluminescence). This light signal is captured by an optical system, and The sequence of these signals is read as the nucleotide sequence. The reaction is carried out in parallel across 200,000 microreactors (for 200,000 overlapping fragments), making it possible to sequence approximately 200 million Base Pairs in 4.5 hours.

Obviously, researchers do not always need to determine the complete sequence of the DNA they are working with. A powerful tool for analyzing complex DNA mixtures for specific sequence elements is Southern blot Hybridization (developed by Edward Southern). The procedure, shown schematically in Fig. 9.7, derives its name from blotting: DNA fragments are separated by gel electrophoresis (initially remaining invisible within the gel), after which a nitrocellulose filter is placed onto the gel. Filter paper is positioned above and below this "sandwich," and the bottom paper layer is immersed in an alkaline solution. Driven by capillary action, the solution migrates upward through the paper layers, "sweeping" the DNA along and transferring it from the gel onto the nitrocellulose membrane. Simultaneously, the DNA is denatured by the alkali. As a result, single-stranded DNA becomes bound to the filter—a medium suitable for subsequent hybridization—making the filter an exact replica of the original gel. The filter is then probed with a radioactively labeled single-stranded DNA fragment of a specific sequence. The probe hybridizes with complementary DNA at specific, previously invisible bands, which can then be visualized by autoradiography.

Fig. 9.7. Blot hybridization

This method can be used, for example, to detect specific DNA sequences in sample preparations; to determine the presence of additional genomic copies of a sequence homologous to a known one; or to identify genes in an uncharacterized genome that are homologous to known genes. One of the numerous Applications of Southern blotting is DNA fingerprinting. The method relies on the presence of minisatellite repeats in Eukaryotic Genomes—small sequence elements that are tandemly repeated multiple times across various genomic loci. The distribution of these loci by repeat number is highly individual, much like a fingerprint. To identify a specific Organism (or person in forensics, legal cases, etc.), genomic DNA is treated with a restriction enzyme that lacks recognition sites within the repeat unit. The resulting fragments are separated by electrophoresis, blotted, and hybridized with a radioactively labeled minisatellite sequence probe. Consequently, the autoradiogram displays a profile of fragments of varying lengths unique to the individual—reflecting different numbers of minisatellite repeats—thus creating a molecular DNA fingerprint.

Northern blotting differs from the Southern blotting procedure described above (the term "northern" being merely a playful analogy derived from Edward Southern's surname) only in that total mRNA extracted from Cells is loaded onto the electrophoresis gel. Hybridization with a labeled DNA probe (such as cDNA from a clone library) makes it possible to detect the presence of a specific mRNA—and thus Gene activity—in a particular Cell type following exposure to activating or repressing factors, as well as to estimate the level of this activity (mRNA concentration) based on the band intensity on the autoradiogram.

Methods based on DNA Microarrays or DNA microchips make it possible to analyze the complete Gene Expression program of an organism or specific cell types under various physiological conditions or during development, as well as to address other questions related to whole-genome functioning.

A DNA fragment up to 1 kb in length with a known genomic Location is amplified, and the single-stranded Amplification products are covalently attached to a small designated area On the surface of a Microscope slide. A 2 × 2 cm Glass slide—a DNA microarray—is patterned with a grid of approximately 6,000 such microspots, each containing DNA from a specific genomic region.

Fig. 9.8. Analysis of total mRNA using a DNA microarray

One of the typical workflows for using a microarray is illustrated in Fig. 9.8. Total mRNA isolated from a specific cell type serves as a template in a reverse METABOLISM/31.html">Transcription reaction. Along with standard NTPs, a fluorescently labeled analog of one of the NTPs is added to the reaction mixture, yielding a preparation of fluorescently cDNAs. Following hybridization with this cDNA, the microarray is scanned using a fluorescence microscope: the presence of a fluorescent spot indicates The activity of a specific gene, while the fluorescence intensity reflects the level of that activity.

Experiments of this type make it possible to uncover shifts in the overall gene expression profile in response to changing environmental conditions, to compare gene activity across different Tissues of a multicellular organism, and to track Changes in the activity of gene groups during cellular differentiation.