Nucleic Acid Sequencing Gels

The amino acid sequence of a protein is determined by identifying amino acid residues as they are sequentially cleaved from the intact protein (see Experiment 2). Sequence analysis of nucleic acids is based on the generation 

---

Polyacrylamide Gel Electrophoresis (PAGE) Nucleic Acid Sequencing Gels 121... 

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). 

As already noted, templated sequencing protocols are now the most generally used methods for nucleic acid sequence determination. The use of the highly processive modified bacteriophage T7 polymerase (Sequen-ase U.S. Biochemical Corp., Cleveland, OH) has eliminated many earlier problems associated with variable intensity of individual bands on sequencing gels. 

Figure 9.11. Comparison of the band separation of linear, wedge, and ionic strength gradient gels. The T reaction of a sequencing experiment carried out on M13 mp8 DNA was run on three different 40 cm 6% polyacrylamide gels, using (a) a standard linear gel, (b) a 0.35-1.05-mm wedge gel, and (c) a 0.05 0.50 Af Tris buffer gradient. [Reprinted, with permission, from A. T. Bankier and B. G. Barrell, in Nucleic Acids Sequencing A Practical Approach , C. J. Howe and E. S. Ward, Eds., Oxford University Press, New York, 1989. IRL Press at Oxford University Press 1989.]...

Many of the techniques used in protein purification procedures have also been adapted for use with nucleic acids. For example, several types of chromatography (e.g., ion-exchange, gel filtration, and affinity) have been used in several stages of nucleic acid purification and in the isolation of individual nucleic acid sequences. Because of its speed, HPLC has replaced many slower chromatographic separation techniques when small samples are involved. 

This technique has been modified to use fluorescently labeled ddNTPs such that each NTP has a specific fluorescent label. The consequence of this change in protocol is that there is no need to use one reaction for each ddNTP. The sequencing reaction can be done in one vial, the separation of sequencing fragments run in one lane, and the terminal dNTP for each fragment identified by the color of the fluorescent label. After the gel is run, the lanes are scanned A/ith an excitation laser, and the fluorescence detected is fed to a computer that records the data and provides a printout of the nucleic acid sequence. 

DNA arrays, like more traditional hybridization techniques such as Southern and Northern blotting, make use of the fact that single-stranded nucleic acid species (DNA and RNA) that possess sequences complementary to each other will hybridize together with exquisite specificity to form double-stranded complexes. In the traditional approaches, the samples to be analyzed are distributed according to size by gel electrophoresis, transferred to and immobilized on solid membrane supports, and probed with specific, labeled complementary nucleic acid sequences. 

Myers, R.M., Fischer, S.G., Maniatis, T. and Lerman, L.S. (1985b) Modification of the melting properties of duplex DNA by attachment of a GC rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3111-3129. 

---