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DNA sequencing with direct blotting electrophoresis and colorimetric detection
Abstract
We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.
IntroductionHistorical Account: Past, Present, and FutureBasic Concepts in Current DNA SequencingDNA Sequencing TechniquesStrategies for Approaching the Target RegionsAutomationStorage, Retrieval, and Interpretation of Sequence Data
Non-radioactive methods to label nucleic acids to be used as probes are being used more often. Non-radioactive detection methods offer several advantages over the usual radioactive methods. Non-radioactive detection methods eliminate the need to deal with the licensing, waste disposal, and safety concerns associated with the use radioactive material. The probes generated are more stable than are probes labeled with 32P. The detection sensitivity of the radioactive and non-radioactive probes is comparable. Non-radioactive detection methods typically require shorter exposure times to detect the hybridization signal [1–5].
DNA sequencing methods, including both Maxam-Gilbert and Sanger di-Principle anddeoxy procedures, have traditionally involved the detection of DNA frag-applicationsments labeled with radioactive isotopes. More recently, other techniques for imaging DNA sequencing ladders with nonisotopic labels have become available. These include colorimetric BCIP and NBT (Richterich et al., 1989), fluorescent (Prober et al., 1988), and chemiluminescent (Beck et al., 1989; Tizard et al., 1990; Creasey et al., 1991; Martin et al., 1991; Bronstein et al., 1992) methods.
The detection of nucleic acids by nonradioactively labeled probes can be performed either with immobilized analytes on blots, by in situ approaches or in solution. The table shows an overview of important application formats including cross reference to the formats described in Chaps. 19–22. With blot and in situ formats, the presence or absence of a particular sequence is recorded, whereas detection in solution allows quantitative measurements. For detection of nucleic acids, a number of blot formats have been established: dot blot, slot blot, Southern blot (DNA analytes), northern blot (RNA analytes), southwestern blot (protein-binding DNA sequences), and genomic blot (analysis of whole genomes). DNA sequencing on blots have also been developed (Richterich et al., 1989; Höltke et al., 1992). A variety of formats have been described for detection oc nucleic acids in situ: colony hybridization (bacterial colonies), plaque hybridization (phage plaques), in situ hybridizations with isolated metaphase chromosomes, tissue sections, biopsies, fixed cells, or whole organisms such as Drosophila embryos. Both proteins and glycoproteins are most often analyzed in western blots. For review of the alternative formats see Matthews and Kricka, 1988; Wilchek and Bayer, 1988; Kessler, 1991; Kricka, 1992.
Binding between analyte and modified probe, resulting in stable hybrid molecules, is most prominently mediated by specific interaction between complementary purine and pyrimidine bases forming A:T and G:C base pairs.
The direct sequencing method represents an efficient and uncomplicated technique to obtain the exact sequence information of PCR [1] products or synthetic PCR standards.
All current state-of-the-art sequencing approaches are based on high-resolution fractionation of single-stranded DNA molecules on Polyacrylamide gel (reviewed in reference 1). Several alternative strategies are being pursued to avoid the use of such gels, but none so far has reached a degree of robustness sufficient to allow routine and reliable sequencing. Among the alternative approaches, the most progress seems to have been made in SBH (Sequencing By Hybridization using, for example, DNA chips coated with large sets of oligonucleotides) (2, 3, 4, 5), but mass spectroscopy (6), scanning tunnelling microscopy (7, 8), single-molecule detection (9, 10), pyrophosphate-release measurement (11) and other approaches continue to be explored.
Here we will describe some important aspects of DNA separation in capillaries that are rather recent and therefore have not been covered in the preceeding chapters. As capillary electrophoresis is a highly instrumental analytical technique, there will obviously be always new important technological developments, as they are described in the first part. However, there are also some interesting new aspects concerning the DNA separation itself, one of them being unique to capillary electrophoresis. They are presented in a second part.
Two common DNA sequencing methods rely on the principle of measuring relative distances of individual nucleotides from a fixed point in a DNA chain (Sanger and Coulson 1975; Maxam and Gilbert 1977). Base-specific sequencing reactions produce nested sets of fragments, sharing one end and terminating at all positions of a given base, for example guanine, in a DNA chain. The ability to determine a sequence is thus dependent on a separation system which is capable of resolving two DNA fragments that differ in length by a single nucleotide. Until very recently, the only system with adequate resolution, suggested in the 1960s, was based on electrophoresis in a flat polyacrylamide gel with a Tris-borate buffer system (Peacock and Dingman 1967). Electrophoresis was conducted between two glass plates in a simple apparatus, based on the design of Studier (1973). After a certain time, controlled by the mobility of marker dyes, the electrophoresis was terminated and the gel was exposed to an X-ray film or to a phosphor-imaging screen to detect DNA bands labelled with 32P, 35S or 33P (batch technique). The nucleotide sequence was then read from the image using band order and specificity of the sequencing reaction (track identity) (Fig. 1A).
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