The base sequence of DNA fragments that have been cloned can be determined. The original procedure for this purpose was the Sanger dideoxy chain termination method illustrated in Figure 34.9. In this method, the single-stranded DNA (ssDNA) to be sequenced is used as the template for DNA synthesis by DNA polymerase (DNA pol). A radiolabeled primer complementary to the 3′-end of the target DNA is added, along with the four deoxyribonucleoside triphosphates (dNTP). The sample is divided into four reaction tubes, and a small amount of one of the four dideoxyribonucleoside triphosphates (ddNTP) is added to each tube. Because it contains no 3′-hydroxyl group, incorporation of a ddNMP terminates elongation at that point. The products of this reaction, then, consist of a mixture of DNA strands of different lengths, each terminating at a specific base. Separation of the various DNA products by size in an electric field using polyacrylamide gel electrophoresis, followed by autoradiography, yields a pattern of bands from which the DNA base sequence can be read. [Note: The shorter the fragment, the farther it travels on the gel, with the shortest fragment representing that which was made first (that is, the 5′-end).] In place of a labeled primer, a mixture of the four ddNTP linked to different fluorescent dyes and in a single reaction tube is now commonly used. The mixture is separated by capillary electrophoresis, the fluorescent labels are detected, and a color readout of the sequence is generated (Fig. 34.10). [Note: The Human Genome Project used variations of this technique to sequence the human genome.] Advances in sequencing technology, so-called next generation, or high-throughput sequencing, now allow the rapid sequencing of an entire genome with increased fidelity and decreased cost through the simultaneous (parallel) sequencing of many DNA pieces. [Note: Sequencing of the exome, that portion of the genome that encodes proteins, is now possible.]
IV. PROBES
Cleavage of large DNA molecules by restriction enzymes produces an enormous array of fragments. How can the DNA sequence of interest be picked out of such a mixture? The answer lies in the use of a probe, a short piece of ssDNA or RNA, labeled with a radioisotope, such as 32P, or with a nonradioactive molecule, such as biotin or a fluorescent dye. The sequence of a probe is complementary to a sequence in the DNA of interest, called the target DNA. Probes are used to identify which band on a gel or which clone in a library contains the target DNA, a process called screening.
A. Hybridization to DNA
The utility of probes hinges on the process of hybridization (or annealing) in which a probe containing a complementary sequence binds a single-stranded sequence of a target DNA. ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane. The immobilized DNA strands are prevented from self-annealing but are available for hybridization to the exogenous, radiolabeled, single-stranded probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the membrane.
B. Synthetic oligonucleotide probes