Life abounds with genetic variations writ in sequences that are often only a few hundred nucleotides long. Rapid detection of these variations for identification of genetic diseases, pathogens and organisms has become the mainstay of molecular science and medicine. This report describes a new, highly informative closed-tube polymerase chain reaction (PCR) strategy for analysis of both known and unknown sequence variations. It combines efficient quantitative amplification of single-stranded DNA targets through LATE-PCR with sets of Lights-On/Lights-Off probes that hybridize to their target sequences over a broad temperature range. Contiguous pairs of Lights-On/Lights-Off probes of the same fluorescent color are used to scan hundreds of nucleotides for the presence of mutations. Sets of probes in different colors can be combined in the same tube to analyze even longer single-stranded targets. Each set of hybridized Lights-On/Lights-Off probes generates a composite fluorescent contour, which is mathematically converted to a sequence-specific fluorescent signature. The versatility and broad utility of this new technology is illustrated in this report by characterization of variant sequences in three different DNA targets: the rpoB gene of Mycobacterium tuberculosis, a sequence in the mitochondrial cytochrome C oxidase subunit 1 gene of nematodes and the V3 hypervariable region of the bacterial 16 s ribosomal RNA gene. We anticipate widespread use of these technologies for diagnostics, species identification and basic research.

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.

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It is thought that changes in mitochondrial DNA are associated with many degenerative diseases, including Alzheimer's and diabetes. Much of the evidence, however, depends on correlating disease states with changing levels of heteroplasmy within populations of mitochondrial genomes, rather than individual mitochondrial genomes. Thus these measurements are likely to either overestimate the extent of heteroplasmy due to technical artifacts, or underestimate the actual level of heteroplasmy because only the most abundant changes are observable. In contrast, Single Molecule (SM) LATE-PCR analysis achieves efficient amplification of single-stranded amplicons from single target molecules. The product molecules, in turn, can be accurately sequenced using a convenient Dilute-‘N’-Go protocol, as shown here. Using these novel technologies we have rigorously analyzed levels of mitochondrial genome heteroplasmy found in single hair shafts of healthy adult individuals. Two of the single molecule sequences (7% of the samples) were found to contain mutations. Most of the mtDNA sequence changes, however, were due to the presence of laboratory contaminants. Amplification and sequencing errors did not result in mis-identification of mutations. We conclude that SM-LATE-PCR in combination with Dilute-‘N’-Go Sequencing are convenient technologies for detecting infrequent mutations in mitochondrial genomes, provided great care is taken to control and document contamination. We plan to use these technologies in the future to look for age, drug, and disease related mitochondrial genome changes in model systems and clinical samples.

Bis(u-acetato)-bis(norbornadiene)dirhodium(I) has been structurally characterized. The compound has been shown to be an active catalyst for the isomerization of quadricyclane to norbornadiene. This material crystallizes in the centrosymmetric monoclinic space group P2j/c with unit cell constants (T = 20 °C) a = 9.7733 (10) A,b = 15.2859 (15) A,c= 12.1815 (14) Á, and ß = 103.563 (8)°. All 3151 unique reflections were used in the X-ray structure analysis which refined to an RF of 0.028. The Rh(l)-Rh(2) distance is 3.1050 (7) A. Each Rh atom is bonded to the two olefin moieties of a norbornadiene ligand and to two oxygen atoms of the bridging acetate ligands. Each Rh coordination sphere has an approximate square-planar configuration with average Rh-olefin and Rh-0 distances of 1.975 (4) and 2.106 (7) Á, respectively. The Rh atoms lie 0.098 and 0.157 Á out of their respective coordination planes. The dihedral angle formed between these planes is 50.1°, while the angle between the planes defined only by the Rh and acetate oxygens is 40.2°. Hydrogen atoms on the norbornadiene groups are within van der Waals contact distances, indicative of strong steric interactions between the ligands of the dimer.Rh(l)-0(2) 2.115 (2) Rh(2)-0(4) 2.104 (2) Rh(l)-0(3) 2.112 (2) Rh(2)-C(8) 2.093 (4) Rh(l)-C(l) 2.104 (3) Rh(2)-C(9) 2.095 (3) Rh(l)-C(2) 2.103 (3) Rh(2)-C(10) 2.088 (3) Rh(l)-C(3) 2.087 (4) Rh(2)-C( 11) 2.096 (3) Rh(l)-C(4) 2.092 (3) Rh(2)-Rh(2) 6.1362 (7) Rh(l)-Rh(l) 4.296 (1) (B) Distances within the Norbornadiene Groups C(3)-C(4) 1.384 (5) C( 1)-C(2) 1.403 (5) C(l)-C(5) 1.531 (5) C(2)-C(6) 1.545 (5) C(3)-C(5) 1.528 (5) C(4)-C(6) 1.545 (5) C(6)-C(7) 1.543 (6) C(5)-C(7) 1.545 (5) C(l)-H(5) 0.93 (4) C(2)-H(8) 0.95 (5) C(3)-H(6) 0.75 (5) C(4)-H(7) 0.94 (5) C(5)-H(4) 1.00 (5) C(6)-H(3) 1.04 (4) C(7)-H(l) 1.02 (5) C(7)-H(2) 0.86 (5) C(2)-C(4) 2.350 (5) C(l)-C(3) 2.322 (5) C(8)-C(9) 1.406 (5) C(10)-C(ll) 1.394 (5) C(8)-C(12) 1.538 (5) C(9)-C(13) 1.537 (5) C(10)-C(12) 1.538 (5) C( 11)-C(13) 1.535 (5) C(12)-C(14) 1.541 (5) C(13)-C(14) 1.546 (5) C(8)-H(15) 0.97 (5) C(9)-H(16) 1.06 (4) C(10)-H(17) 0.94 (5) C(ll)-H(18) 0.96(5) C(12)-H(19) 0.98(5) C(13)-H(20) 0.93 (4) C(14)-H(21) 1.04 (5) C(14)-H(22) 1.01 (5) C(8)-C(10) 2.335 (5) C(9)-C(l 1) 2.345 (5) 0(2)-Rh(l)• C(4)-Rh(l)-C(3)-Rh(l)-C(3)-Rh(l)-C(l)-Rh(l)-C(l)-Rh(l)-C(2)-Rh(l> C(3)-Rh( 1 )-C(4)-Rh(l)-C(l)-Rh(l). C(2)-Rh(l)-C(4)-Rh(l)-C(3)-Rh(l)-