Amplification of Hsp90 homologs from plant-parasitic nematodes using degenerate primers and ramped annealing PCR.

Procedures for using PCR to amplify novel members of gene families from genomic DNA frequently involve the use of degenerate primers. Of key importance is the ability to produce ample quantities of specific PCR product while minimizing or eliminating nonspecific side reactions. Primer design protocols and software are improving, but finding the optimal amplification conditions often involves a tremendous amount of time and expense. Degenerate PCR primers can be designed based on consecutive stretches of conserved amino acids. General criteria for guiding this process have been described (5,8), and a number of standalone or Web-based computer programs exist to facilitate primer selection (9,10). A comparison of sequences from closely related organisms, combined with knowledge of codon usage in the target organism, is enormously helpful. Selected homologous protein sequences are typically aligned with programs such as ClustalW (12). Stretches of consecutive amino acids that are identical in all of the aligned sequences are chosen for reverse translation, and the corresponding nucleotide sequences are used in the design of consensus or degenerate oligonucleotide primers. A direct alignment of nucleic acid sequences can be used if sufficient nucleotide sequence homology exists. The use of degenerate rather than consensus primers in PCR increases the possibility of including a primer with exact complementarity to an unknown target DNA sequence. However, in highly degenerate primer pools (> 500-fold), the actual concentration of an exact match primer will be a very small fraction of the total added. Unfortunately, there are no hard-and-fast rules regarding an upper limit on primer degeneracy, and the success of any primer pair must be empirically determined. Innumerable failures have occurred when the primers were either incorrect “guessmers”, yielding no products, or too degenerate, giving rise to a multitude of confusing, nonspecific products. One solution to this problem is to design more than one set of degenerate primers to the same site, thereby keeping substitutions to a minimum in each primer pool. Another solution is to substitute inosine for N, for its ability to base pair with any nucleotide. Improvements in the cycling parameters have received less attention, but a few methods, such as hot start PCR (4), have achieved wide acceptance. PCR optimization typically involves titration of template, primer, dNTPs, Taq DNA polymerase, and various buffer additives. The choice of optimum annealing temperature is particularly important. In conventional PCR, an annealing temperature of 5°C less than the predicted melting temperature of the primers is generally recommended. This advice is often inadequate for degenerate primers, which contain a mixture of primer sequences with highly variable annealing optima. The hallmark of touchdown PCR (6,7) is that the annealing temperature is lowered 1°C every two cycles, thereby allowing the most specific PCR products to gain a head start over nonspecific amplification products. Stepdown PCR (7) simplifies this program somewhat by lowering the annealing temperature in fewer but sharper increments. We have modified these approaches further in what we call ramped annealing PCR (RAN-PCR) in which the annealing temperature is gradually reduced within each PCR cycle. This modification led to the successful amplification of heat shock protein (Hsp90) genes from nematode genomic DNA using degenerate primers and may be generally applicable for degenerate PCR on complex, uncharacterized templates. Nematode genomic DNA was prepared according to Bird and Riddle (3). Genomic DNA from the pine wood nematode (Bursaphelenchus xylophilus) was the generous gift of Robert I. Bolla (St. Louis University, St. Louis, MO, USA). Genomic DNA extracts were prepared from single adult lesion nematodes by the method of Thomas et al. (11). PCRs were conducted in 20-μL total volume and contained 10 mM TrisHCl, pH 8.3, 50 mM KCl, 3 mM MgCl2, 0.1% Triton X-100, 0.005% gelatine, 200 μM dNTPs, 25 ng genomic DNA, 1 U DisplayTAQ (PGC Scientific, Gaithersburg, MD, USA), and 0.5 μM each primer, U831 [5′-AA(T/C) AA(A/G)AC(A/C )AAGCC(A/C/ G/T)T(T/C)TGGAC-3′] and L1110 [5′TC(A/G)CA(A/G)TT(G/A/C)TCCATGAT(A/G)AA(G/A/C) AC-3′]. Primers were synthesized by Sigma-Genosys (Woodlands, TX, USA). Degeneracy is 64-fold for U831 and 72-fold for L1110. Primers were evaluated for duplex and hairpin formation using Oligo Version 4 (Molecular Biology Insights, Cascade, CO, USA) (9). Hot-start reactions were assembled as follows: 10× DisplayTAQ buffer, MgCl2, dNTPs, primers, and water were combined in the bottom of a microcentrifuge tube and topped with a drop (30–50 μL) of melted paraffin wax (Walnut Hill, Bristol, PA, USA). After the wax cooled, forming an even barrier, a top layer of additional buffer, water, template, and Taq DNA polymerase was added. RAN-PCR consisted of a preliminary denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 20 s, 65°C for 20 s, 60°C for 5 s, 55°C for 5 s, 50°C for 5 s, 45°C for 15 s, and extension at 72°C for 1 min, ending with a 5-min final extension at 72°C. RANPCRs were conducted in 0.5-mL thin wall microcentrifuge tubes in a PTC130 with the Hot Bonnet attachment (MJ Research, Watertown, MA, USA). Degenerate primers were designed based largely on the known Caenorhabidits elegans Hsp90 sequence and both gradient PCR and touchdown PCR amplified specific Hsp90 products from C. elegans DNA (not shown). However, attempts to use the same conditions to amplify Hsp90 using DNA from our “test” plant parasite, the soybean cyst nematode, were unsuccessful (not shown). This led us to develop our modification of the touchdown procedure, RAN-PCR. In this method, annealing temperature is gradually reduced within each amplification cycle rather than between cycles. The initial annealing temperature was set at 65°C, a condition that favors reaction specificity. The temperature was reduced in 5°C increments Benchmarks