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mismatches, although Yokota notes that it may recognize A typical hybridization strategy uses short pieces of DNA, some incorrect combinations more easily than others. Later, perhaps 15–25 nucleotides each. That means cutting large the repair crew arrives. MutL binds to the MutS–DNA com- genes (10,000–100,000 base pairs) into numerous pieces plex, MutH cleaves the mismatched and checking every one. Sun and strand, and a helicase enzyme removes Yokota, on the other hand, take an the unwanted DNA. “aerial” view with AFM, looking at In Sun and Yokota’s method, MutS much larger fragments in less detail. is a solo act. Four single-stranded “AFM has a favorite size of DNA, 449-nucleotides-long fragments of [which is] several hundred base pairs,” DNA are mixed and matched, and Yokota says. “[But] it can go up to MutS is added. Then the mixture is 10,000 base pairs.” spread on an atomically flat sheet of This approach could allow researchmica, a process that straightens the ers to screen DNA fragments, perhaps Atomic force microscope image of (a) a sinDNA and makes it easy to see where revealing regions where mismatches gle and (b) a double MutS–DNA complex. proteins are attached using AFM (see are clustered or absent, before a more Anal. Chem. 1999, 71, 83 A; 1663– detailed microarray analysis. In exper1667; 4418–4422). The researchers iments conducted so far, Sun and use the microscope’s tapping mode to Yokota report that the measured posiminimize the damage to the sample. “Maybe [the tip] tions of the MutS–DNA complexes agree well with the contacts the DNA for a brief period of time,” Yokota says, predicted locations of the mismatches, and two complexes “but it’s gentle enough so that it doesn’t damage the DNA separated by only 45 base pairs could still be resolved. or the protein.” The approach is quite new, and some improvements Of course, there are other ways to locate DNA mismatch- could be made. One possibility might be to use a variety es, including sequencing and hybridization techniques. In of MutS proteins, giving each a special assignment. Right the latter approach, the 5´ strand of a “normal” DNA senow, the researchers use only E. coli’s MutS. “[But] many quence is hybridized to the 3´ strand of sample DNA, and bacteria produce their own versions of MutS with slight mismatches are identified using electrochemical or optical differences,” explains Yokota. “Some may be better at dedetection. A more sophisticated version is used in DNA mitecting G/C mismatches; others, A/T mismatches; and so croarrays—a popular SNP-detection strategy. on.” This approach might enhance the accuracy of the “The DNA chip hybridization method has very good method and provide specificity at the same time. A lot resuccess, but there are gaps,” Yokota says. “That’s the reamains to be seen, but nature may have just given us anson my technique may be useful.” other valuable hint. Elizabeth Zubritsky
Human genome sequenced On June 26, the Human Genome Project
institutions worldwide—and Celera have
accuracy of 99.9%. About half of the
consortium and Celera Genomics an-
been working concurrently using differ-
genome is in “near-finished form,”
nounced the completion of their respec-
ent sequencing strategies, and they
and just under one-quarter is com-
tive “working drafts” of the human
have announced slightly different re-
pletely finished, according to a state-
genome. These drafts contain the se-
sults. The consortium reports
quences for large fragments of DNA,
that its efforts have
tional Human Genome
spanning all human chromosomes, in the
covered 97% of the
Research Institute.
proper order.
human genome
Celera reports
with an average
that >99% of the
The public consortium—a group of 16
514 A
A N A LY T I C A L C H E M I S T R Y / A U G U S T 1 , 2 0 0 0
ment released by the U.S. Na-
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Test tubes without walls
S. NILSSON
The Lund researchers used the new system to examine lipolysis (breakdown of lipids) in adipocytes (fat cells). The Several analytical techniques for monitoring chemical events addition of isoprenaline, a -adrenergic agonist, was shown and intracellular processes at the single-cell level are now avail- to stimulate lipolysis, leading to the release of fatty acids and able. Such analyses are quite popular in the area of drug disa decrease in pH. Changes in pH could be easily followed covery because of their ability to detect small cellular changes by adding a pH-dependent fluorophore to the droplet and induced by pharmaceuticals. A big problem associated with continually monitoring changes in fluorescence with a CCD these small-scale analyses (volumes
