Amplification of gene fragments with very high G/C content: c7dGTP and the problem of visualizing the amplification products.

Abstract

Nieders~chsisches Institut ffir Peptid-Forschung GmbH, D-30625 Hannover, Germany PCR amplification of sequences from genomic DNA often yields multiple nonspecific bands, especially if the amplified regions are rich in guanosine and cytosine. G/C-rich sequences can cause hairpin loop structures that potentially give rise to problems for in vitro DNA amplification. In addition to this, G/Crich primers are less specific because of a shorter stretch needed for recognition. Inclusion of the nucleotide analog 7'deaza-2'-deoxyguanosine-5'-triphosphate (cTdGTP) in the PCR reaction is the most suitable strategy that allows efficient amplification of the expected sequences. (1~ First of all, PCR conditions should be optimized concerning the choice of primers, concentrations of all components, and cycling parameters. The primers used are the most critical parameter, determining success or failure of an amplification reaction. (2~ Sometimes it is not possible to choose optimal primers, for example, because of a high G/C content of the investigated region. In this case, one should consider the application of c7dGTP. The base analog 7'-deaza-2'-deoxyguanosine precludes Hoogsteen bond formation, as the N-7 of the guanine ring is replaced by a methine. (1~ The stability of Watson-Crick base-pairing is also reduced (AH: dG-dC base pair: 5 0 kJ/ mole of stack/c7dG-dC:-36 kJ/mole of stack. (3} Therefore, c7dGTP reduces tertiary structure formation within the fragments and causes longer hybridizing stretches necessary for the chosen annealing temperature. This enhances specificity of the PCR. It has been shown that c7dGTP can fully replace dGTP during PCR-amplification. (4} However, the usage of c7dGTP confronts the investigator with another problem: Fragments containing this analog can be stained only poorly with ethidium bromide. Normally, ethidium bromide fluorescence is enhanced if it intercalates into double-stranded DNA. The incorporation of c7dGTP impairs ethidium bromide binding in the following way: The electron pair of N-7 purine nitrogens within the major groove of the DNA interacts with ethidium bromide. Because c7dGTP lacks this nitrogen, the binding of ethidium bromide is reduced. Moreover, the decrease of N-7 nitrogen changes the dipole moment of the nucleotide, which alters adjacent base stacking and consequently the intercalation of ethidium bromide. (4} Therefore, the detection of DNA fragments containing c7dGTP upon gel electrophoresis is difficult when using ethidium bromide staining. We tested silver staining as a method for visualizing those DNA fragments.