PubMed Health⌕ Search

Biomedical subjects

J G Nadeau

Publications and source records attributed to J G Nadeau.

5 recordsLinked to original sources
PubMed1992

Strand displacement amplification--an isothermal, in vitro DNA amplification technique.

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.

Base Sequence↗
PubMed1992

Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.

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 single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, 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 enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.

Base Sequence↗
PubMed1989

Structural basis for DNA bending.

We report proton NMR studies on DNA oligonucleotides that contain A tracts of lengths known to produce various degrees of bending. Spectra of duplexes in the series 5'-(GGCAnCGG).(CCGTnGCC) (n = 3, 4, 5, 7, 9) reveal substantial structural changes within the An.Tn tract as its length is increased. Chemical-shift comparisons show that A tracts with fewer than about seven members do not contain regions of uniform [or poly(dA).poly(dT)-like] structure. Long An tracts (n greater than or equal to 7) appear to consist of an internal segment of homopolymeric conformation flanked by regions of transitional structure that occupy about four A.T pairs on the 5' side and two A.T pairs perhaps the directly adjacent G.C pair on the 3' side. In shorter duplexes (n less than 7), these two transitional regions overlap and an apparent mutual incompatibility causes length-dependent changes that are most pronounced near the 3' end. Throughout the series, there is a striking monotonic relationship between the location of an A.T pair in the A tract and the relative position of its ThyH3 resonance. The direction of the chemical-shift dispersion is opposite to that expected from consideration of ring-current effects alone; this discrepancy suggests a gradual decrease in ThyH3...N1Ade hydrogen-bond length as one moves from the 5' to the 3' end of the A tract and from short to long A tracts. Nuclear Overhauser effect measurements reveal that the interproton distances AdeH2...H1'Ade and AdeH2...H1'Thy vary along each A tract, except in the central regions of the longer ones where they are fairly constant and in good agreement with the poly(dA).poly(dT) structure proposed by Lipanov, A.A. & Chuprina, V. P. [(1987) Nucleic Acids Res. 15, 5833-5844]. This model features a substantial negative base-pair tilt, which has been suggested previously as the source of A-tract bending. In contrast, the nuclear Overhauser effect distances are inconsistent with at least one known crystallographic A-tract structure [DiGabriele, A. D., Sanderson, M. R. & Steitz, T. A. (1989) Proc. Natl. Acad. Sci. USA 86, 1816-1820], which lacks appreciable base-pair tilt.

DNA↗
PubMed1985

Anomalous hairpin formation in an oligodeoxyribonucleotide.

An accurate method for deriving molar absorptivity-temperature profiles applied to a set of single-stranded oligodeoxyribonucleotides shows that the undecamer CGAGTTTGACGp exists in a hairpin conformation involving Watson-Crick base pairing between the two terminal CG dinucleotides. The hairpin, which has a transition midpoint of 40 degrees C in 0.115 M Na+, is unusually stable in comparison with previously reported hairpins. A non-linear least squares analysis of the undecamer's profile in terms of a two-state equilibrium model indicates that the hairpin-to-coil transition occurs with an enthalpy change about twice that expected if only combinations of Watson-Crick base-paired stacking interactions are considered. The analogous hairpin structure (containing an identical CG/CG stem) assignable to the complementary strand CGTCAAACTCGp does not form above 0 degrees C. Measurements on the two undecamers indicate that variation in non Watson-Crick interactions within the loops of two similar hairpins can produce a difference in stability of at least 2.2 kcal/mol (25 degrees C, 0.115 M Na+), roughly equal to the amount contributed to a double helix by a 5'-CG-3'/5'-CG-3' base-paired stacking interaction.

Base Composition↗