Marker/sequence links

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

Alternative protocols for covalent coupling were described by Wu et al. [48], Sims et al. [49], and McKie et al. [50]. In the latter approach, single-stranded DNA was linked in a multistep procedure with a short primer covalently coupled to the antibody. The single-stranded DNA-marker also included a Hind III restriction site. By adding the restriction enzyme previous to the PCR-step, the DNA-marker sequence was released from the immobilized immuno-complex to the supernatant liquid phase and subsequently transferred to capillary vessels (see Section 2.2.3). 

Restriction enzymes, sequence-dependent cleavage of DNA by, 12 497—498 Restriction fragment linked polymorphism (RFLP), 12 500 procedure, 12 103-104 Restriction sites, as genetic markers, 12 500... 

These results are simply explained in the patterns of Figure 9 in the compact complex, the marker (M) is wedged in between the polymer sequences and its mobility is low. In the gel-like structure the mobility of the marker is not affected, because the marker is mainly surrounded by sovlent molecules since the cross-linking between macromolecules is very low. Polarized luminescence results lead to confirmation of the complex structure proposed from visco-metry studies. 

Access to nucleic acid dendrimers is initiated by a zip-fastener like dissociation of the DNA double strand by heating. The double strand separates into the two individual strands by thermal motion (denaturation). Subsequent association, hybridisation of complementary sequences, is followed by stepwise cross-linking to form DNA dendrimers, which can contain up to two million oligonucleotide-end group strands (Fig. 8.19). The latter can be labelled with fluorescence or radioactive markers. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

In the multiplex-IPCR assays carried out by Joerger and Hendrickson, DNA of different lengths was used for separation of the PCR amplificates (see Fig. 4). Results of their experiments are discussed below in Section 3.5. The ability to discriminate between different DNA markers by length is nevertheless limited by the separation capabilities of the gel. For a large number of different DNA probes, a sequence-specific detection carried out, for example, by PCR-enzyme-linked oligonucleotide sorbent assay (ELOSA Section 2.2.2) should be preferable. 

We have studied the sequence determinants for helical hairpin formation during the insertion of a model membrane protein into the ER membrane. To simplify the problem, we engineered a 40-residue long poly(Leu) stretch into a membrane protein that inserts readily into ER-derived microsomes when expressed in vitro (Fig. 2A). Asn-X-Thr acceptor sites for N-linked glycosylation were used as topological markers, as they can only be modified when located in the... 

Evidence for the link between PKC activation and cell proliferation was initially provided by the demonstration that two intracellular events associated with cell replication - a rise in cytosolic pH and the expression of the proto-oncogenes c-fi/s and c-tnyc - are controlled by PKC PKC stimulates the membrane bound Na /H exchange mechanism in smooth muscle which extrudes intracellular H in exchange for extracellular Na this leads to a rise in intracellular pH, a prerequisite for cellular DNA replication (Mitsuka and Berk, 1991). c-fbs and c-myc are proto-oncogenes whose transcription to mRNA is one of the earliest markers of cell proliferation they encode for proteins, found in the cell nucleus, which initiate the sequence of events leading to DNA synthesis (Rozen-gurt, 1991). 

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