News
Genetic screening with FENs Using flap endonucleases (FENs), Mary Ann D. Brow and colleagues at Third Wave Technologies have found a new way to identify single-base mutations in DNA. Unlike restriction endonucleases, FENs don't recognize specific sequences of doublestranded DNA. Instead, they recognize and cleave "flaps"—extraneous single-stranded sequences that hang off a double-stranded structure. Flaps are created when a singlestranded oligonucleotide (oligo) displaces another, essentially taking over its "turf in the double-stranded structure. The FENs in this study cleaved the flaps that resulted when an upstream oligo displaced the 5' end of a downstream oligo. The researchers found that the site of cleavage depended on the extent of the two oligos' overlap. Primer extension of the upstream oligo was not required, meaning that flaps could be created by adding pairs of overlapping oligo probes targeted to a region of interest in the DNA. Cleavage of the downstream probe would then indicate the presence of the target sequence.
NMR and the purple fragments
terium salinarium. They have diameters ranging from 0.2 to 2 um and are negatively charged wiih a high bacteriorhodopEmploying what has become a standard sin content Unlike liquid crystalline me"bag of tricks", NMR spectrometrists have dia, there is no critical lower threshold become adept at collecting data on large concentration required for using the memmolecules drat can be used for determining brane fragments structural and for alignment. dynamic information. One such The NIH scientrick is aligning tists tested the biomolecules in membrane fragsolution relative ment approach to the external with two 15N-lamagnetic field to beled proteins— measure onethe Va domain of bond internuclear die human T-cell dipolar couplings. receptor and ubiqThese couplings uitin, a well-studied are easily meabiomolecule. Signifsured if the mo15 1 N- H one-bond dipolar couplings measured icant alignment lecular alignwas seen with die for 57 backbone amides of ubiquitin versus ments are weak. Va domain protein, values calculated for the eolution structure. Liquid crystalline and a best fit bemedia have been used successfully to imtween the observed dipolar couplings and pose this type of alignment, but many proa preliminary X-ray structure showed teins destructively interfere with the most good agreement With ubiquitin it was popular liquid crystalline phases. Ad Bax necessary to add at least 50 mM NaCl to and colleagues at the National Institutes of weaken the electrostatic interactions Health (NIH) demonstrate that a suspen(which caused line broadening) between sion of planar purple memhrane fragments itself and die purple membrane fraecan also be used for alioning marrnmolements The resultant 15 N-'H dipolar coucules in a strong magnetic field. plincrc agree with the values calculated
To quantify the results, the researchers developed a microplate assay. The wells of a microplate were coated with steptavidin. After being cleaved, the flap hybridized to a biotinylated DNA template, and DNA polyThe purple membrane fragments are merase added digoxigenin-labeled nucleotieasily isolated and purified from Halobacdes. The resulting duplex was bound to the streptavidin label and detected using chemifluorescence. Because uncleaved probes or probes broken at sites other than the cleavproduced a signal for both probes. A simiage site were not labeled, the signal was pro- lar test was conducted with the wild-type portional to the concentration of target DNA CFTR gene and the AF508 mutation, which causes cystic fibrosis. (Nat. BiotechTo identify mismatches, the researchnol. 1999,77, 292-96) ers took advantage of the fact that a single base pair mismatch one base upstream of the cleavage site prevented cleavage. To screen for the Leiden mutation, the most common risk factor for thrombosis, a wildtype probe and a mutated probe were synthesized. Homozygous wild-type DNA samples produced a signal only with the wildtype probe homozygous mutant samples proDetection of the earry gene ffom the euman cytomegalovirus duced a signal only with (hCMV). After the 5 "ffap" of the eignal probe is cleaved, it ,inds to the mutated probe and the template and is labeled with hU residues bearing digoxigenin. heterozygous samples (Adapted with permission. Copyright 1999 Nature America.) 306 A
Analytical Chemistry News & Features, May 1, 1999
from a hp^f fit of thp solutinn
