Any DNA that has been cloned requires a sequencing step for confirmation. Though this can now be performed using sequencers and fluorescently labeled nucleotides, such a technique cannot be used in certain experiments like promoter mapping, where the sequencing reaction has to be run in the same gel with the cDNA.
Theoretically, a sequencing reaction is very simple and requires the use of dideoxynucleotides and a thermostable enzyme; however, when it comes to practical technicalities, it is really very difficult to get a proper and readable gel. I have found that the use of Promega’s fmol Cycle Sequencing System helps in standardizing sequencing reactions and provides all the ingredients required for performing them.
The material provided includes the thermostable DNA Polymerase, from Thermus aquaticus, required for the reaction, which is carried out using a thermal cycler. This is a general PCR reaction, the difference being that dideoxynucleotides are used to randomly terminate elongation. This results in a sequence ladder that is resolved by denaturing polyacrylamide gel electrophoresis. Because radioactivity is employed, the reaction is very sensitive and the sequence can be read from a radiogram after exposure to X-ray film or by phosphorimaging. All four nucleotides are provided along with the dideoxynucleotides in a ready-to-use form. The ratio of these has already been optimized for almost all kinds of templates.
The kit can be used for GC rich templates as well; it contains deaza-nucleotide mixes that help in resolving bands in GC-rich regions. I have used this reaction mixture for a GC-rich template (approximately 65% GC) for promoter mapping and it worked extremely well; no optimization was required.
End-labeling of sequencing primers using gamma-labeled ATP is recommended and the materials required, such as T4 Polynucleotide Kinase and its buffer, are provided. Only the radioactivity has to be supplied; the kit offers the choice of using gamma P32, P33 or S35 labeled ATP. The labeled primers can be used directly in the sequencing reaction without further purification, although I find it wise to at least check the primer after labeling by running an aliquot on a denaturing polyacrylamide gel.
A very small amount of starting DNA (in the range of femtomoles) is required. Both PCR product and plasmid DNA can be used as templates. However, the DNA should be of high quality to make the reaction work efficiently. After the completion of the PCR, the reaction is stopped by adding the stop solution provided in the kit. The reaction can then be loaded onto a sequencing gel. The stop solution is colored and contains glycerol that helps in loading the samples.
Using this kit, I have sequenced PCR products as well as the cloned region in plasmids. Templates were prepared using either Novagen’s SpinPrep™ PCR Clean-up Kit or Sigma’s GenElute Plasmid Miniprep Kit. I have used this kit for sequencing two templates at the same time. However, more samples can be processed at the same time. Since I have used radioactivity for the labeling of primers, the reaction becomes highly sensitive and a small amount of sample is required for one run. The remaining sample can be used later if required. I run the reaction on a 0.3 mm Urea-PAGE prepared for sequencing. The gel is dried after run and is phosphor imaged. Reading of 150 bases was sufficient in my experiment. I have, however, read a sequences up to 250 bases in one lane. Multiple lanes can be run depending upon the length to be read.
The kit also contains primers and DNA templates for control reactions. A descriptive handbook is also provided that has information about the procedure and a trouble shooting guide in case the reaction does not work.
I am completely satisfied with the performance of the kit. Since the kit requires the use of radioactivity, precautions must be taken while handling the reactions.