2. Characterizing and Using Cloned DNA Fragments

7. EXPERIMENTAL FIGURE 9-21 Gel electrophoresis separates DNA molecules of different lengths. A gel is prepared by pouring a liquid containing either melted agarose or unpolymerized acrylamide between two glass plates a few millimeters apart. As the agarose solidifies or the acrylamide polymerizes into polyacrylamide, a gel matrix (orange ovals) forms consisting of long, tangled chains of polymers. The dimensions of the interconnecting channels, or pores, depend on the concentration of the agarose or acrylamide used to form the gel. The separated bands can be visualized by autoradiography (if the fragments are radiolabeled) or by addition of a fluorescent dye (e.g., ethidium bromide) that binds to DNA.

8. The dimensions of the interconnecting channels, or pores, depend on the concentration of the agarose or acrylamide used to form the gel. The separated bands can be visualized by autoradiography (if the fragments are radiolabeled) or by addition of a fluorescent dye (e.g., ethidium bromide) that binds to DNA.

12. Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method

15. Characterizing and Using Cloned DNA Fragments

17. Characterizing and Using Cloned DNA Fragments Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method (a) A single (template) strand of the DNA to be sequenced (blue letters) is hybridized to a synthetic deoxyribonucleotide primer (black letters). The primer is elongated in a reaction mixture containing the four normal deoxyribo nucleoside triphosphates plus a relatively small amount of one of the four dideoxyribo nucleoside triphosphates. In this example, ddGTP (yellow) is present. Because of the relatively low concentration of ddGTP, incorporation of a ddGTP, and thus chain termination, occurs at a given position in the sequence only about 1 percent of the time. Eventually the reaction mixture will contain a mixture of prematurely terminated (truncated) daughter fragments ending at every occurrence of ddGTP. EXPERIMENTAL FIGURE 9-23 Cloned DNAs can be sequenced by the Sanger method, using fluorescent tagged dideoxyribonucleoside triphosphates (ddNTPs).

19. Characterizing and Using Cloned DNA Fragments Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method FIGURE 9-23(b) To obtain the complete sequence of a template DNA, four separate reactions are performed, each with a different dideoxyribonucleoside triphosphate (ddNTP). The ddNTP that terminates each truncated fragment can be identified by use of ddNTPs tagged with four different fluorescent dyes (indicated by colored highlights).

21. Characterizing and Using Cloned DNA Fragments Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method (c) In an automated sequencing machine, the four reaction mixtures are subjected to gel electrophoresis and the order of appearance of each of the four different fluorescent dyes at the end of the gel is recorded. Shown here is a sample printout from an automated sequencer from which the sequence of the original template DNA can be read directly. N nucleotide that cannot be assigned. [Part (c) from Griffiths et al., Figure 14-27.]

24. The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture