DNA and RNA sequencing is an essential technique that is mandated for DNA cloning, characterization, mutagenesis, DNA recombination and gene expression.1-3 Very recently, nucleic acid sequencing has become a rapid and powerful tool to identify novel genes targeted by a novel drug or protein. To discover new target molecules, one may not need to utilize traditional gene or cDNA cloning methodologies nor does the isolated gene or cDNA need to be completely sequenced. Instead, as long as a portion of exon sequences is sequenced, a potentially novel DNA molecule can be identified by computer database searching. Nevertheless, nucleic acid sequencing is a crucial tool for one to fulfill such a task. How does one sequence the DNA of interest? How does one identify the existence of mRNA expression in a cell or tissue type of interest? Which methodology is the best selection for one's purposes? The present chapter summarizes the author's extensive experience and will help one achieve one's goals.

There are several well-established methods for nucleic acid sequencing. Based on the author's years of hands-on sequencing experience, this chapter describes in detail the protocols for DNA or RNA sequencing by nonisotopic or isotopic sequencing approaches. Specific methodologies include nonisotopic and isotopic DNA sequencing by dideoxynucleotides chain termination,23 formamide gel sequencing,34 primer walking,5 unidirectional deletions,6 direct sequencing by PCR and RNA sequencing27-9 with modifications. These methods have been successfully employed in our laboratories. Although automatic fluorescent sequencing is a very attractive methodology, because of the high cost of the DNA sequencer, we do not consider this method to be within the scope of the present chapter, which is designed for regular nucleic acid sequencing in modestly equipped laboratories.

Since Sanger et al.2 developed the dideoxynucleotides chain termination method of DNA sequencing, it has been extensively modified by the use of some superior enzymes and DNA cloning vectors. The general principles of the method include: (1) a synthesized oligonucleotide primer anneals to the 3' end of the DNA template to be sequenced; (2) a DNA polymerase catalyzes the in vitro synthesis (5' 0 3') of a new DNA strand complementary to the template, starting from the primer site, using deoxynucleoside 5'-triphosphates (dNTPs). One of the dNTPs is biotinylated dATP or dCTP used for nonisotopic sequencing, or a-35SdATP or a-35SdCTP utilized for isotopic sequencing; and (3) following the synthesis of the new DNA strand, it is terminated by the incorporation of a nucleotide analog that is an appropriate 2',3'-dideoxynucleotide 5'-triphosphate (ddNTP). All four ddNTPs lack the 3'-OH group required for DNA chain elongation. Based on the nucleotide bases of the DNA template, one of the four ddNTPs in each reaction is incorporated. The enzyme-catalyzed polymerization will then be stopped at each site where a ddNTP is incorporated, generating a population of chains of different sizes. Therefore, by setting up four separate reactions, each with a different ddNTP, complete nucleotide sequence information of the DNA strand will be revealed.

In DNA or RNA sequencing, different factors and strategies should be considered. The following factors greatly influence nucleic acid sequencing.

1. Quality of template. The purity and integrity of DNA or RNA to be sequenced are essential for obtaining an accurate and complete nucleotide sequence. Make sure that the DNA or RNA template is not degraded prior to sequencing. The DNA template can be single-stranded DNA from M13 cloning vectors, double-stranded plasmid DNA or double-stranded bacteriophage DNA with appropriate pretreatment. mRNA template is usually transcribed into cDNA followed by standard DNA sequencing.

2. DNA polymerase. United States Biochemical (USB) and Amersham Life Science produce excellent DNA polymerase for nucleic acid sequencing. These include Sequenase version 2.0 DNA polymerase and AMV reverse transcriptase, which are routinely used in our laboratory with successful results. Sequenase version 2.0 DNA polymerase is a superior enzyme that has no 3' ^ 5' exonuclease activity. These enzymes should be kept at -20°C prior to use.

3. Primers. For sequencing the region of the DNA template close to the cloning site of the vector, primers are complementary to the vector DNA strands and are commercially available. For the sequence beyond 250 to 500 nucleotides from the cloning site, new primers should be designed based on the nucleotide sequence information obtained from the very last sequencing. The quality of primers is critical for the success of DNA or RNA sequencing. Primers can be designed by computer programs such as Oligo version 4.0 or CPrimer f. We routinely check with the Tm value, potential intrahairpin structures, potential interloop formation and GC contents of the designed primers before employing them in sequencing.

4. Nonisotopic or isotopic dNTP. Biotinylated dATP or dCTP is commonly used for nonisotopic DNA or RNA sequencing. The drawback is potential background due to the high sensitivity. Nonetheless, the nonisotopic approach is faster and safer compared with radioactive sequencing. In the near future, traditional isotopic methods may be completely replaced by nonisotopic methodology. 32P-labeled dNTP (dATP or dCTP) has a high energy level and a short half-life (usually 14 days). A major disadvantage of using 32P is that it gives diffuse bands on autoradiographic x-ray film, limiting readable information of DNA sequences. However, [32S]dATP has a lower energy level and a longer half-life (usually 84 to 90 days) and greatly improves autoradiographic resolution. Therefore, [32S]dATP is recommended for isotopic DNA or RNA sequencing. The activity and amount of labeled nucleotide play a significant role in nucleic acid sequencing.

5. Compressions. Compressions represent a common problem in DNA sequencing, which is primarily due to dG-dC rich regions that may not be fully denatured during electrophoresis. As a result, interruption of the normal pattern of migration of DNA fragments becomes obvious. The bands are usually spaced closer than usual (compressed together) or occur further apart than usual, resulting in a significant loss of sequence information. In order to solve this problem, we use dITP or 7-deaza-dGTP (USB) to replace the nucleotide dGTP, which forms a weaker secondary structure that can be readily denatured during electrophoresis. In addition, a formamide gel is helpful in this compression situation. With these modifications, we have found that bands are sharper and compressions eliminated by the use of dITP and formamide. In some cases, some bands appear weak using both dGTP and dITP. This limitation can be eliminated using pyrophosphatase (USB) in the presence of Mg2+, Mn2+, or both. The manganese can allow one to obtain sequence information close to the primer site.

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