G. Terrance Walker

Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.

In SituStrand Displacement Amplification

In situamplification of nucleic acids has provided a means to analyze for the presence of single copy DNA (single copy genes or viral infection) and expression of low abundance RNAs. To date, PCR has been used extensively(1) but nonetheless presents some significant technical challenges that can be difficult to overcome. In particular, the thermal cycling of cells and tissues is generally detrimental to cell morphology and tissue integrity and can limit the success of the procedure and the efficacy of the results. To circumvent this issuein situamplification of nucleic acids can be accomplished using an isothermal amplification method such as strand displacement amplification (SDA) (2) The basic procedure involves cellular fixation/permeabilization followed by amplification which proceeds at a constant moderate temperature for a short period of time followed by the detection of amplicon. As an example of the effects of temperature on cellular integrity, peripheral blood mononuclear cells (PBMCs) can be analyzed by measuring forward and side scatter using fluorescence activated cell sorting (FACS) to differentiate among monocytes, lymphocytes and granulocytes. This differentiation is retained after exposure of the PBMCs to an SDA temperature profile, but is abolished after exposure to thermal cycling (personal communication, Becton Dickinson, Immunocytometry Systems). Furthermore, specific cell surface epitopes are retained after an SDA thermal profile as demonstrated by FACS analysis of cell surface antigens.

Enhanced Signal Generation by Target Amplification

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. The basic reaction is comprised of three steps: 1. Denaturation of the target DNA 2. Annealing of sequence specific primers 3. Template-specific elongation of these primers with a DNA polymerase and desoxynucleotides