Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two singlestranded target DNA fragments (T, and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of PI binds to the 3' end of T,, forming a duplex with 5' overhangs.Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction Tuberculosis is one of the most common human infectious diseases, infecting an estimated one billion worldwide with =.16 million active cases and 3 million deaths per year (9). Recently, it has received renewed attention due in part to its high incidence among AIDS patients. The disease is caused by Mycobacterium tuberculosis and Mycobacterium bovis. Culture-based diagnosis provides exquisite sensitivity and specificity but requires 3-6 weeks due to the slow growth rate of most pathogenic species of mycobacteria. Acid-fast organisms can be quickly identified in stained smears but with low sensitivity (>104 organisms per ml). Consequently, there is an acute need for a rapid and sensitive test.The PCR meets these diagnostic requirements and has been applied to mycobacterial infections (10). An IS6110 insertion element, which is specific for M. tuberculosis and M. bovis, provides an effective target sequence for amplification and detection (11). SDA was applied to genomic DNA samples from M. tuberculosis and M. bovis using a portion of the IS6110 element as target sequence. We have achieved a 106-fold amplification, thereby establishing SDA as an isothermal alternative for amplifying specific DNA sequences. MATERIALS AND METHODS

The equilibrium binding of ethidium to the right-handed (B) and left-handed (Z) forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) was investigated by optical and phase partition techniques. Ethidium binds to the polynucleotides in a noncooperative manner under B-form conditions, in sharp contrast to highly cooperative binding under Z-form conditions. Correlation of binding isotherms with circular dichroism (CD) data indicates that the cooperative binding of ethidium under Z-form conditions is associated with a sequential conversion of the polymer from a left-handed to a right-handed conformation. Determination of bound drug concentrations by various titration techniques and the measurement of circular dichroism spectra have enabled us to calculate the number of base pairs of left-handed DNA that adopt a right-handed conformation for each bound drug; 3-4 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to the right-handed form for each bound ethidium, while approximately 25 and 7 base pairs switch conformations for each bound ethidium in complexes with poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2, respectively. The induced ellipticity at 320 nm for the ethidium-poly(dG-dC).poly(dG-dC) complex in 4.4 M NaCl indicates that the right-handed regions are nearly saturated with ethidium even though the overall level of saturation is very low. The circular dichroism data indicate that ethidium intercalates to form a right-handed-bound drug region, even at low r values where the CD spectra show that the majority of the polymer is in a left-handed conformation.