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PCR, on balance When PCR is used to compare genomic DNA samples, the preferential amplification of fragments from one sample and the interference caused by stray impurities can be problems. Now, G. Mike Makrigiorgos and colleagues at Harvard Medical School present a technique called balanced PCR that they say removes the bias from PCR and retains the quantitative relationship between two DNA samples. In balanced PCR, a target genome is digested with a restriction enzyme, and the resulting DNA fragments are ligated to a composite linker sequence (LN1). LN1 contains a short tag that marks fragments as genomic DNA. Similarly, a control genome is digested and ligated to another composite linker sequence (LN2), which marks fragments as control DNA. LN1 and LN2 also contain one common sequence, P1, so that a primer that hybridizes to P1 will recognize both LN1 and LN2 fragments. The ligated target and control DNA samples are then mixed, and PCR is
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Relative expression levels for 30 genes in lung and prostate cDNA samples (a) before and after balanced PCR and (b) before and after conventional PCR. (Adapted with permission. Copyright 2002 Nature America.)
performed on the blended sample using the P1 primer. Because the P1 primer does not discriminate between target and control DNA, there is no preferential amplification of one sample, and the relative proportions are maintained. Because the target and control DNA are mixed, there is less chance that stray impurities will impede one reaction. After amplification, the target and control DNA samples can be identified by the unique tags within LN1 and LN2 and subse-
Raman looks at carbon electrodes
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quently separated. After testing the approach on several simple samples, the researchers used balanced PCR in the screening of lung and prostate cDNA on microarrays. When the relative expression levels were calculated for 30 genes, there was less distortion using balanced PCR than conventional PCR. The researchers also verified that up to three rounds of balanced PCR amplification and separation can be performed with good results. (Nat. Biotechnol. 2002, 20, 936–939)
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Researchers interested in examining electrochemical interfaces in situ often turn to surface-enhanced Raman spectroscopy, but carbon electrodes do not support electromagnetic field enhancement.
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To circumvent this problem, Richard McCreery at Ohio State University and Takashi Itoh at Tohoku University (Japan) use Raman spectroscopy with very sensitive charge-coupled device detectors
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to study 4-nitroazobenzene (NAB) chemisorbed on a glassy carbon electrode in an electrochemical cell. The researchers say that this is the first report of in situ Raman spectra for monolayers on carbon without electromagnetic field enhancement. They propose that a chemisorbed NAB monolayer on a glassy carbon electrode changes structure as the monolayer
The reaction that accompanies electron transfer from glassy carbon to NAB.
switches to a high conduction state. Unusual interactions between
lution during electrochemical reaction, and the researchers sug-
NAB and the electrode’s π system were found by comparing the
gest that such interactions may occur with other aromatic mono-
chemisorbed NAB–carbon electrode surface with NAB in the so-
layers. (J. Am. Chem. Soc. 2002, 124, 10,894–10,902)
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