TUCSON, ARIZONA
University of Arizona

Allele-specific PCR in maize
--Natividad, M; Winkler, RG

Allele-specific PCR is a commonly applied method for the speedy detection of known single-base polymorphisms in DNA utilizing specially designed oligonucleotides. It requires the design of a specific oligonucleotide that selectively amplifies one allele over another. Specificity is achieved by designing the oligonucleotide to match the desired allele but mismatch other alleles near the 3' end of the allele-specific oligonucleotide. The mismatch between the DNA and the oligonucleotide results in specific amplification of the desired allele and little or no amplification of the undesired alleles by preventing elongation at the 3' end by the enzyme.

We and others have found that designing allele-specific oligonucleotides is not trivial. Our work has involved designing allele-specific oligonucleotides for the GC-rich d3 gene in maize. The gene's high GC content makes it inherently predisposed to generate non-specific PCR product. Amplification of the gene produced non-specific banding, making it necessary for us to optimize. After having considered the numerous methods published for increasing the specificity of PCR products, we have realized a new approach to obtaining specific PCR product. We have successfully optimized for the desired point mutation by varying the oligonucleotide, MgCl2, DNA, and enzyme concentrations simultaneously.

There have been numerous reports suggesting ways of enhancing the specificity of allele-specific PCR:

It has been found that the use of dimethylsulfoxide (DMSO) (Winship et al., Nucl Acids Res 17:1266, 1989) and nonionic detergents (Bachmann et al., Nucl Acids Res 18:1309, 1990) improves the DNA sequencing reaction possibly by decreasing inter- and intrastrand reannealing. DMSO has also been found to improve amplification of the retinoblastoma gene (Bookstein et al., Nucl Acids Res 18:1666, 1990). However, DMSO has also been found to reduce DNA synthesis by Taq polymerase by 50% in PCR assays (Gelfand and White, in PCR Protocols--A Guide to Methods and Applications, p. 137, 1990).

Tetramethylammonium chloride (TMAC) is another reagent that is proven to increase PCR specificity (Hung et al., Nucl Acids Res 18:4953, 1990). TMAC has been found to reduce potential DNA/RNA mismatch. Titration studies showed that TMAC used at concentrations of 1X10-4 M - 1X10-5M can effectively eliminate non-specific amplification without any inhibitory effects on Taq polymerase.

The absence of KCl has been reported to be optimal for the amplification of DNA molecules in the range of 3-6 kbp (Ponce et al., Nucl Acids Res 20:623, 1991).

E.coli single-stranded DNA binding protein (SSB) has been found to facilitate the amplification of genomic sequences by PCR (Oshma, BioTechniques 13:188, 1992). Its stability at temperatures of up to 100 C makes it suitable for use in PCR (Weiner et al., J Biol Chem 25:1972-1980, 1975).

The use of formamide has been reported to improve specificity of PCR products particularly in GC rich genes (Sarkar et al., Nucl Acids Res 18:7465, 1990).

It has also been found that increasing the denaturation temperature for GC rich genes improves specificity (Dutton et al., Nucl Acids Res 21:2953-2954, 1993).

A "touchdown" method for decreasing annealing temperatures improves specificity for some genes (Don et al., Nucl Acids Res 19:4008, 1991).

It has been reported that diluting DNA 10-fold of the standard genomic DNA concentration, lowering MgCl2 concentration below 1.5 mM, and lowering the amount of enzyme to 0.2-0.3 U/25 µl can increase specificity of PCR product (Bottema et al., Meth Enzymol 218:388-402, 1994).

All PCR reactions were performed in a final volume of 50 µL containing PCR buffer (50 mM Tris pH 9.0, 250 mM KCl, 0.5% gelatin, 0.5% Triton X-100) (Triton X-100 was later eliminated since it inhibited transfer to membrane; the elimination of Triton X-100 had no effect on the results of the experiments). Original 1X concentrations from which optimizations were performed are as follows: 50 µM dNTP's; 200 ng DNA; 1 µM oligonucleotide; and 0.5 U enzyme /50 µL reaction. PCR reactions were contained in 0.2 mL thin walled tubes. PCR was conducted using a PTC-100 HB Programmable Thermal Controller (MJ Research, Watertown, MA, USA) with the following parameters: initial denaturation at 95 C for 1 min.; and 35 cycles of denaturation at 94 C for 30 s; annealing at 60 C for 30 s; and extension at 72 C for 2 min. PCR products were electrophoresed on 2% IBI Agarose (Eastman Chemical Company, New Haven, CT, USA) in 1X TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) and analyzed by ethidium staining. PCR product was confirmed by dot blot.

Specific PCR product for the GC rich d3 gene was achieved with DNA (1X), 0.3 mM MgCl2, 0.25X oligonucleotide concentration, and 0.5X enzyme concentration. Lowering the DNA concentration by one tenth its original concentration did not produce product. Lowering MgCl2, oligonucleotide, and enzyme improved specificity, however exceedingly low concentrations did not produce product. Optimizing for each reagent in combination showed some improvement over optimizing for each reagent individually. However, when the conditions were optimized in combination, the effects were not additive or synergistic on increasing specificity.

Initial experiments proved that specificity of PCR product was increased when reagent concentrations were lowered. After optimizing for each reagent and still encountering non specificity, we decided to optimize the reagents simultaneously. Under the assumption that optimizing the reagents would produce specific PCR product, we were surprised to find no additive or synergistic effect on improving specificity. This suggests that optimizing DNA, MgCl2, oligonucleotide, and enzyme concentrations contribute the same specificity effect possibly through the same or similar mechanisms. In future endeavors we would concentrate on identifying a component that attacks a different mechanism to achieve specific PCR product.

Figure 1. Optimization table used to achieve specific PCR product. A) DNA and MgCl2 were simultaneously optimized with 1X enzyme in order to identify specific PCR product. B) The optimized DNA and MgCl2 concentrations were then used to optimize for the proper oligonucleotide and enzyme concentrations that would produce specific PCR product. +++ denotes that product was present and specific; +/- denotes that product was present but not specific; - denotes that product was not present. Original 1X concentrations from which optimizations were performed were as follows: 50 µM dNTP's; 200 ng DNA; 1 µM oligonucleotide; 0.5 U enzyme/50 µL reaction.


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