DNA Cloning: The Basics:- Specific DNA Sequences Are Detectable by Hybridization
DNA hybridization, a process outlined in Chapter 8 (see Fig. 8–32), is the most common sequence-based process for detecting a particular gene or segment of nucleic acid. There are many variations of the basic method, most making use of a labeled (such as radioactive) DNA or RNA fragment, known as a probe, complementary to the DNA being sought. In one classic approach to detect a particular DNA sequence within a DNA library (a collection of DNA clones), nitrocellulose paper is pressed onto an agar plate containing many individual bacterial colonies from the library, each colony with a different recombinant DNA. Some cells from each colony adhere to the paper, forming a replica of the plate. The paper is treated with alkali to disrupt the cells and denature the DNA within, which remains bound to the region of the paper around the colony from which it came. Added radioactive DNA probe anneals only to its complementary DNA. After any unannealed probe DNA is washed away, the hybridized DNA can be detected by autoradiography (Fig. 9–9).
FIGURE 9–9 Use of hybridization to identify a clone with a particular DNA segment. The radioactive DNA probe hybridizes to complementary DNA and is revealed by autoradiography. Once the labeled colonies have been identified, the corresponding colonies on the original agar plate can be used as a source of cloned DNA for further study.
A common limiting step in detecting and cloning a gene is the generation of a complementary strand of nucleic acid to use as a probe. The origin of a probe de pends on what is known about the gene under investigation. Sometimes a homologous gene cloned from another species makes a suitable probe. Or, if the protein product of a gene has been purified, probes can be designed and synthesized by working backward from the amino acid sequence, deducing the DNA sequence that would code for it (Fig. 9–10). Now, researchers typically obtain the necessary DNA sequence information from sequence databases that detail the structure of millions of genes from a wide range of organisms.
