Fluorescein-labeled primers

The question is similar to asking how many times one can strip and reuse a microarray before performance deteriorates. An alternative approach is provided by Hessner et al. (2003a) in which the cDNA probes are permanently labeled using fluorescein-labeled primers to the clone s vector insert region. Fluorescein is excited at 488 nm and emits at 508 nm, while Cy3 may be excited at 543 nm to reduce any spectral overlap with fluorescein. Thus, fluorescein-labeled cDNA probes may be printed down and the slide scanned for QC/QA purposes prior to hybridization. Since the same region is primer-labeled in each cDNA, a direct comparison between the relahve fluorescence units (RFUs) and the amount of cDNA probe can be defermined. 

Figure 7.11 CE-LIF separation and quantitation of fluorescein-labeled lambda PCR product. A 500 bp product was generated with titrated amounts of lambda bacteriophage template and primers, one of which was fluorescein-labeled. The fragment was analyzed by CE-LIF, and only the fiuor from the PCR product was detected. With increasing amounts of DNA template, peak height and area of the product also increased up to a point, whereupon the PCR plateaued. Note the increase in primer and primer-dimer peaks as template availability decreases. (Reproduced with permission from KJ Ulfelder, Applications Information Bulletin A-1774, 1994. Copyright Beckman Instruments, Inc.)...

Real-time hybridization of 5 -fluorescein-labelled target oligonucleotides was described using a fibre-optic DNA sensor by Ehrat [32], Lu [33] and coworkers, and a fibre-optic DNA sensor array capable of positively identifying a point mutation of a biotin-primer-labelled PCR product was reported by Walt [34]. 

Specific fluoresceine isothiocyanate (FTTC) or Cy5-labeled primers designed according to the DNA-sequence of the wild-type strain. 

T4 RNA ligase can introduce fluorescent 3 -O-(5 -phos-phoryldeoxycytidyl) S-bimane phosphorothioate into RNA or DNA (Cosstick et al., 1984). Other methods have been developed for fluorochrome labeling of DNA for automated sequencing, either by labeled primers or dye-terminators (fluorescent ddNTP Applied Biosystems) with DNA polymerase or with terminal dNTP transferase (Trainor and Jensen, 1988). We successfully used fluorescein-dUTP (Boehringer Mannheim, soon available), both for automated sequencing and probe preparation. All common DNA polymerases incorporate this analogue efficiently. 

The labeling procedure, based on established random primer technology (3) can be used with DNA of various types and purity see Chapter 15). The resulting fluorescein-labeled probes can be stored for several months, thus avoiding frequent labeling reactions. Valuable probe is not lost since a further purification step before hybridization is not required. 

Labeled primers Oligonucleotide primers are easily labeled on the 5 -phosphate group with biotin, digoxygenin, dinitrophenol, other haptens, fluorescein, other fluorophores, and many other reagents. These labels can be used for either capture or detection. To increase specificity, the primer label is generally used for capture, rather than for detection. Thus, hybridization to a labeled oligonucleotide probe can be performed in solution where kinetics are more rapid, and followed by capture on the solid phase. 

When unlabelled 5 -primer is used for DNA amplification, the PCR products are labelled with fluorescein following the protocol explained in next section (Section 37.4.2). 

Figure 37.2 shows the results obtained with PCR products. Figure 37.2a shows the PCR products detection on 1.2% agarose gel. Figure 37.2b and 37.2c displays the analytical signals obtained with genosensor device for different DNA copies of the PCR products, which have been labelled using the FITC labelled 5 -primer (Fig. 37.2b), and those obtained for PCR products which have been labelled using the fluorescein ULS kit (Fig. 37.2c). When the PCR products are labelled using the FITC labelled...

Fig. 1. Single copy gene detection. 1, 2, and S pg of an coRI digest of human genomic DNA blotted onto Hybond-N. Hybridized with a 1.5-kb probe for N-ras proto-oncogene labeled with fluorescein-dUTP by the random primer labeling reaction see Chapter 15). Hybridization with 10 ng/mL of probe, overnight, at 60 C 30-min exposure on Hyperfilm-MP.

In contrast to the deoxynucleotide counterpart, TDTase incorporates preferentially and almost exclusively a single fluorescein- or biotin-riboUTP at the 3 terminus of oligonucleotides (57). The nonisotopically labeled oligonucleotides (or primers) are suitable for use in nucleic acid hybridization, DNA sequencing, and PCR priming. 

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