PCR labeling

For PCR labeling of Ml3, follow the directions in Step 3 except modify the deoxynucleotide triphosphate mix to include three unlabeled nucleotides and one labeled nucleotide (yielding a final concentration for each at 10 pM). Ten cycles of amplification should suffice. 

Esposito DL, Palmirotta R, Veri MC, Mammarella S, D Amico F, Curia MC et al (1998) Optimized PCR labeling in mutational and microsatellite analysis. Clin Chem 44 1381-1387... 

PCR labeling very simple and powerful labeling method, small quantities of high specific activity can be rapidly obtained, does not require cloned templates for probe synthesis, primers (and their sequence) should be known... 

Fig. 1. The effect of digoxigenin-ll-dUTP on mol wt and amplification efficiency in PCR labeling. Amplification products were electrophoresed on a 1% agarose minigel and stained with ethidium bromide. The first luie contains molecular markers (I-kbp ladder Gibco BRL, Gaithersburg, MD). The second lane contains the target plasmid amplified without digoxigenin. and the third lane the plasmid amplified with 10% of the dTTP replaced by dioxigenin-11-dUTP. This high level of dUTP was used to show both the mol-wt shift and decreased amplification efficiency associated with higher levels of probe modification.

Figure 5.6 Determination of base composition by multiple PCR labeling reactions. indicates stable-isotope labeling. Reproduced with permission from Ref [92). 1999 American Chemical Society.

Figure 6. cDNAs amplification products hybridized with a DlG-labeled 742 bp probe. Lane 1, 2,4 and 6 apple pectin culture medium, lanes 3, 5 and 7 glucose culture medium. Lanes 1, 2 and 3 to isolate ri3 and lanes 4, 5, 6 and 7 to isolate t2- Lane S corresponds to the hibridation with the 742 bp probe cloned in the vector PCR TM (In-vitrogen) digested with Eco RI and lane 9 A digested with Pstl. 

A site at the Agricultural Experimental Station (Ithaca, NY) was treated in microcosms with C-labeled glucose, phenol, caffeine, and naphthalene. Levels of C02 were measured to assess utilization of the substrates, and the populations analyzed by separating the C-labeled DNA by density centrifugation, followed by PCR amplification and sequencing of 16S rRNA (Padmanabhan et al. 2003). Populations contained relatives to a range of bacteria that varied with the substrate. Only relatives of Acinetobacter were found in all samples, and for caffeine only Pantoea. 

The STR data generated at NIST were based on amplifying i.o ng of the genomic DNA with fluorescent labelled primers. The PCR amplified products were analyzed by slab gel electrophoresis followed by imaging with a Molecular Dynamics Fluorl-mager 595 or by capUlary electrophoresis using a PE-ABI 310 Genetic Analyzer. 

Since the MHLW designated shrimp/prawn and crab for mandatory labeling in June 2008, respective PCR methods to discriminate between shrimp/prawn and crabs in processed foods have been developed. 

Shoji A, Hasegawa T, Kuwahara M et al (2007) Chemico-enzymatic synthesis of a new fluorescent-labeled DNA by PCR with a thymidine nucleotide analogue bearing an acridone derivative. Bioorg Med Chem Lett 17 776-779... 

Fig. 19.1 Differential displays comparing RNAs from saline (S)-, imipramine (I)- or fluoxetine (F)-treated rats. Total RNA was extracted from hypothalami of animals treated with the different drugs for two months. Autoradiograms of amplified -[35S]-dATP-labeled PCR (polymerase chain reaction) products after electrophoresis in 6% polyacrylamide gels are shown for two different primer combinations that identified one upregulated (arrowhead) and one downregulated (arrow) fragment in the groups treated with antidepressants (from [4] with permission).

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

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