In addition to cloning and amplifying DNA, molecular techniques are used to analyze DNA molecules through a determination of their sequences and an investigation of their functions.
DNA sequencing A powerful technique to emerge from recombinant DNA technology is the ability to quickly sequence DNA molecules. DNA sequencing is the determination of the sequence of bases in DNA. Sequencing allows the genetic information in DNA to be read, providing an enormous amount of information about gene structure and function. Details of DNA sequencing will be covered in Chapter 19.
In situ hybridization DNA probes can be used to determine the chromosomal location of a gene or the cellular location of an mRNA in a process called in situ hybridization. The name is derived from the fact that DNA or RNA is visualized while it is in the cell (in situ). This technique requires that the cells be fixed and the chromosomes be spread on a microscope slide. The chromosomes are then briefly exposed to a solution with high pH, which disrupts the pairing of the DNA bases, making them accessible to probes. A labeled probe, which binds to any complementary DNA sequences, is added. Excess probe is washed off, and the location of the bound probe is detected. Originally, probes were radioactively labeled and detected with autoradiography, but now many probes carry attached fluorescent dyes that can be seen directly with the microscope (iFigure 18.19a) Several probes with different colored dyes can be used simultaneously to investigate different sequences or chromosomes.
In situ hybridization can also be used to determine the tissue distribution of specific mRNA molecules, serving as a source of insight into how gene expression differs among cell types (I Figure 18.19b). A labeled DNA or RNA probe complementary to a specific mRNA molecule is added to tissue, and the location of the probe is determined by using either autoradiography or fluorescent tags.
DNA footprinting Many important DNA sequences serve as binding sites for proteins; for example, consensus sequences in promoters are often binding sites for transcription factors (see p. 000 in Chapter 11). A technique called DNA footprinting can be used to determine which DNA sequences are bound by such proteins.
In a typical DNA-footprinting experiment, purified DNA fragments are labeled at one end with a radioactive isotope of phosphorus, 32P. An enzyme or chemical that
118.19 In in situ hybridization, DNA probes are used to determine the cellular or chromosomal location of a gene. (a) Fluorescent probes are used to mark the locations of specific gene sequences on chromosomes. (b) In situ hybridization can also be used to detect specific mRNA sequences in tissues. Here, the distribution of mRNA produced by the ftz gene (which has a role in controlling development) in a young Drosophila embryo is detected by a radioactive probe and autoradiography. [Autoradiograph from E. Hafen, A. Kuriowa, and W. J. Gehring, Cell 37:833-841.]
Regions bound by RNA polymerase _
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