Biotinylated PCR products

Biotinylated PCR products For purposes of nonradioactive detection, a single biotin molecule is incorporated into the 5 end of one or both of the PCR primer pairs. The biotinylated PCR products are used in a chemiluminescent detection system as described below. Attachment of biotin molecules to oligonucleotides has been described in detail (12). For an alternative method of biotinylating PCR products, see Note 3. 

The amplification reaction is carried out in IX PCR buffer containing 100 yM dNTPs, 30 pmol of biotinylated primers, 10-100 ng of human template DNA, and 2 U of Taq polymerase. The PCR profile consists of an initial denaturation at 95°C for 5 min. The samples are then subjected to PCR with 30-s incubations at 94, 55, and 72 C for 35 cycles. See Note 3 for an alternative method of biotinylating PCR products. A lengthy discussion of optimization of PCR reactions is beyond the scope of this chapter, but further information can be found in Note 5 and references given therein. 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

A disposable electrochemical enzyme-amplified genosensor was described for specific detection of Salmonella (Del Giallo et al., 2005). A DNA probe specific for Salmonella was immobilized onto screen-printed carbon electrodes and allowed to hybridize with a biotinylated PCR-amplified product of Salmonella. The hybridization reaction was detected using streptavidin conjugated-AP where the enzyme catalyzed the conversion of electroinactive a-naphthyl phosphate to electroactive a-naphthol, which was detected by differential pulse voltammetry. 

The genosensors used for the PCR products detection are streptavidin-modified SPCEs with biotinylated probes as sensing phase [1,2]. 

ECL has also been applied to the analysis of polymerase chain reaction (PCR) products [53,55], PCR is first used to amplify the specific genes by use of two primers, one of which is biotinylated. The double-stranded DNA is then captured on streptavidin-coated magnetic beads and washed with an alkaline solution to denature and separate the strands. The particle-bound, single-strand DNA is used to capture products hybridized with Ru-labeled complementary... 

Fig. 8.4. Biosensors may become interesting for probe detection. In this example (from Olson et al., 1991), a biotinylated and a fluoresceinated probe are hybridized in solution to the target (I). The primer sequences should be located outside this region for PCR-product detection. Avidin acts as a bridge between the biotinylated probe and the biotinylated surface of the biosensor (II). Fluorescein is detected with a urease-antibody conjugate. The rise in pH, after the addition of urea, is detected with the pH-sensitive biosensor. The total assay time is less than 2 h with a detectability of 30 amol and a coefficient of variation (standard deviation/mean) of less than 10%.

This protocol is based on simplex assays however, multiplexing with np to 3 internal primers can be performed, either from the same PCR product or different PCR prodncts. The primer design software can only determine one internal primer at a time, often the first choice primers for each will not be useful in a multiplex assay where the combined seqnence to analyze is best designed to generate unique SNP dispensations. In addition, the orientation of the primers is vital for multiplex assays as only one PCR primer can be biotinylated. 

PCR-ELISA is a hybrid assay combining PCR as a first step and using ELISA as a detection system for the amplicons (Figure 9.1). The PCR step produces biotinylated amplicons that are bound further to a streptavidin-coated microtiter plate. The amplicons are denatured to produce single-stranded PCR products, and only the biotinylated strand stays in the well of the microtiter plate. A FITC-Iabeled probe is bound to the PCR strand, a step that adds an extra level of specificity to the assay. An enzyme-labeled anti-FITC antibody is used to produce a colorimetric signal that can be measured by an ELISA reader. This method has been applied successfully to the detection of <10 ppm hazelnut in processed foods and food ingredients, which eliminated the potential for cross-reactivity observed in ELISA (Holzhauser et al., 2002). 

Figure 6. Allele Quantitation ofAPOE PCR Product with ddCTP and biotinylated ddUTP. Two PCR products were prepared, one from a heterozygote and one from a homozygote at site 112, a T/C polymorphic site in the ApoUpoprotein E gene. The two PCR products were mixed in a 1 5 heterozygote homozygote ratio and typed with a genotyping primer of sequence 5 -TGGGCGCGGACATGGAGACC-3 with a mixture of ddCTP and biotinylated...

Figure 7. Allele Quantitation with Modified ddNTP. Mixtures of ratios of homozygous and heterozygous PCR products for the APOE E locus were prepared and genotyped with ddCTP and ddTTP (regular Sequazyme-PinPoint assay), or a mixture ofddCTP and biotinylated ddUTP. The measured fraction of A allele, as assayed by the peak area offraction ofddT or biotinylated ddU incorporation, was plotted vj. the known fraction of the A allele in the...

Biotinylation of PCR products can be accomplished by two methods attachment of biotin to the 5 ends of the PCR primers as described in this chapter (12), or by direct incorporation of the nucleotide precursor, bio-ll-dUTP, into the PCR product during amplification (15,17). If direct incorporation of biotin into the amplitied DNA is desired, use the nucleotide analog such that its final concentration is 10- to 20-fold less than the normal precursor, TTP. In this way, a bio-dUTP will be added every tenth to twentieth base where normally a TTP would be incorporated. For example, a 200-bp PCR product with 50 thymidine bases in its sequence, will contain an average of 5 to 2.5 biotins, and this amount is more than sufficient for detection purposes by the chemiluminescent method. The biotin nucleotide analog may be obtained from Enzo Diagnostics (New York, NY), Bethesda Research Laboratories (Gaithersburg, MD) or other reliable sources. 

Figure 5.13 MALDI-TOF mass spectrum of a Sanger sequencing ladder generated by primer extension from PCR products using a mixture of normal elongators (dNTPs) and biotinylated terminator nucleotides (ddNTPs). The termination products are purified on a streptavidin-coated solid support. The sequence of the target region is...

Bettazzi et al. used an electrochemical low-density DNA array in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens. Cor a 1.04 and Cor a 1.03, in foodstuff. Unmodified PCR products were captured at the electrode interface via sandwich hybridization with surface-tethered probes and biotinylated signaling probes. The resulting biotinylated hybrids were coupled to a streptavidin-alkaline phosphatase conjugate and then exposed to an a-naphthyl phosphate solution. Differential pulse voltammetry (DPV) was used to detect the a-naphthol signal with detection limits as 0.3 and 0.1 nmol for Cor a 1.03 and Cor 1.04, respectively. The results are comparable with the ones obtained with classical ELISA tests. 

---