PCR Product

PCR can also be used to modify DNA sequences using primers differing at one or several positions from the target sequence. This is possible because PCR does not require perfect complementarity of a primer to the sequence flanking the target. Since all of the PCR products contain the primer sequence, an insertion or deletion can thus be incorporated into the product by modifying a primer. It is also possible to add new sequences to the 5 -ends of the primers. Modified or additional genetic information may thus be multiplied and transr ported. 

Quantitative polymerase chain reaction, also called real-time RT-PCR or QPCR, is a method which employs insertion of a signal, such as fluorescence or enzyme activity, into PCR products generated by RT-PCR to determine the amount of messenger RNA (mRNA) in a tissue accurately. 

The first DNA preparations in this part of the study was PCR product - DNA of Chlamydia trachomatis 17 > bp), in the presence of a smaller by molecular mass internal control of human DNA. After migration the gel was exposured for 5, 30, 300 and 600 seconds by transilluminator Vilber Lourmat, equipped with 6 UV lamps with irradiance W = 0,24 W/m2 and 254 nm filter. The degree of structural integrity loss of amplificated DNA was evaluated by the decrease of brightness intensity of the of the bands processed by using the tools of "ImageJ" computer program. 

Specific allergenic ingredient Western blot or PCR kits Target protein or gene Relative molecular weight (Da) or PCR product length (bp)... 

Because the PCR exponentially copies the target molecule or molecules, amplicon contamination in the laboratory is a serious concern. It is recommended that the mastermix is prepared in an isolated area, such as a PCR station equipped with a UV light. This work area should be exposed to UV radiation after use to destroy any DNA contaminants. The use of dedicated pipets and Altered pipet tips is also recommended. The template DNA should be prepared and added to the reaction in an area that is isolated from the mastermix preparation hood. The thermal cycling and gel electrophoresis should be conducted in a third work area and care should be taken not to introduce amplified PCR products into the mastermix or template preparation work areas. 

The plant sample in lane 6 is also positive for the transgene of interest. Because the band for the effect gene (middle band) is typically fainter than the band for the selectable marker gene (bottom band), it appears that for lane 6, the PCR product amplitication for the effect gene is below the assay detection threshold. Because the selectable marker is clearly present and the PCR amplitication worked, lane 6 can be interpreted as a positive result for the transgene of interest. 

Figure 20 Plot of PCR products produced against the number of amplification cycles. (A) Theoretical PCR product amplified and (B) actual PCR product amplified...

Figure 22 Real-time quantitation of PCR products. The straight line represents the threshold fluorescence value. Each curved line is a plot of the PCR products formed against the number of cycles for different samples. For samples containing 100% GMO, only B cycles are required to reach the threshold fluorescence. Samples containing 0.01% GMO will require F cycles before the threshold is attained...

Ulfelder, K., Anderson, K., and Schwartz, H. E., Analysis of PCR products and DNA restriction fragments to detect AIDS (HIV-1) virus in blood, poster presentation, HPCE 91, San Diego, CA, 1991. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

Figure 4.2. Schematic illustration of directional cloning of PCR products. The sequence 5 CACC is required at the 5 end of the PCR product for directional topoisomerase-mediated cloning. In the example shown, the 5 -CACC sequence is appended immediately 5 of the ATG start codon of the gene to he inserted.

An alternative to repeated cloning of PCR products is a recombination-based approach developed by Liu et al. (1998) to permit the cloning of a PCR product into a plasmid and the rapid conversion of the plasmid to a number of different expression systems without the necessity of cloning the PCR product multiple, independent times. The method, termed the univector plasmid-fusion system (UPS), involves the insertion of the PCR product into a particular type of plasmid, called the univector, which can then be placed under the control of a variety of promoters or fused in-frame to various tag sequences. The system is based upon plasmid fusion using the Cre-lox site-specific recombination system of bacteriophage PI (Sternberg et al., 1981). The Cre enzyme is a site-specific recombinase that catalyzes recombination between two 34 base pair (bp) loxP sequences and is involved in the resolution of dimers formed during replication of the... 

Figure 4.4. Schematic illustration of directional topoisomerase cloning of PCR products into the pUNI vector. The PCR product to be cloned has the sequence 5 -CACC appended at the 5 end to direct the orientation of cloning. The Vaccinia virus topoisomerase I enzyme forms a covalent adduct with the cloning vector to create a cloning competent plasmid construct. The loxP site is 5 to the insertion site. The vector and PCR product are designed to fuse the ORF in-frame with loxP.

Figure 4.6. Recombinational cloning (RC). A. Cloning of PCR products using the attB + attP > attL + attR reaction catalyzed by Int and IHF. The result is an Entry Clone that can be used to create functional vectors. B. Conversion of an Entry Clone to a functional vector using the attL + attR > attB + attP reaction catalyzed by Int, Xis and IHF. A wide variety of functional vectors can be constructed by using a Destination Vector with the appropriate promoters and tags.

Each of the -6000 PCR products was then co-transformed into yeast along with the recipient vector that had been linearized using a restriction enzyme that digests the plasmid at the desired cloning site. The 70 bp of homologous flanking sequence on each end of the PCR products is sufficient for the yeast homologous recombination system to act upon and insert the PCR product into the vector (Hudson et al., 1997 Ma et al., 1987). 

The most efficient strategy may be to use the topoisomerase cloning method in conjunction with the Crs-lox or X recombination systems. In this way, PCR products can be efficiently inserted into the Univector or Entry Clone with minimal additional sequences added to PCR primers. Once the genes are inserted into the starting vectors they can rapidly be converted to functional vectors using either Cre-lox or X recombination. 

Classical PCR involves detection of a PCR product by electophoretic mobility on a gel, which is time consuming. Real-time PCR is distinct from classical PCR, in that electrophoresis is avoided and the PCR product is detected... 

---