Introduction

PCR is a well-known and effective tool for the amplification of DNA targets of interest. When DNA targets high in GC content are amplified, PCR product formation can often be compromised by inadequate strand separation and the propensity for complex secondary structure formation. The inability of the DNA polymerase to negotiate through regions of strong secondary structure, such as hairpins, often results in the formation of truncated PCR products. In addition, mispriming amplification products are prevalent when primers are high in GC content (1). Despite the inherent challenges, detection of GC-rich sequences is becoming increasingly important for the molecular diagnosis of inherited diseases (2).

Traditional approaches, such as specialized DNA polymerases and Hot Start technologies, have the potential to improve some GC-rich PCR assays provided that secondary structure formation is minimal (1). Many unique methods have been developed to enable amplification of sequences high in GC content, including the addition of organic molecules, the modification of thermal cycling protocols, and the use of modified dNTPs. Although each of the above-mentioned methods provides an improvement in GC-rich target amplification, various combinations of these methods are often required to obtain the desired specificity.

One of the most noteworthy modified dNTPs used to amplify GC-rich targets is 7-deaza-dGTP, a dGTP analog that lacks nitrogen at the 7 position of the purine ring. The use of this modified nucleotide blocks Hoogsteen bond formation without interfering with normal Watson-Crick base pairing, reducing the probability that secondary structures, such as G quadruplexes, will form (1). To further improve GC-rich target amplification, a Hot Start version of 7-deaza-dGTP has recently been developed. This new dNTP analog is an elegant fusion of the secondary structure reducing nucleotide analog 7-deaza-dGTP and TriLink BioTechnologies' CleanAmp™ dNTP Hot Start technology. The CleanAmp™ dNTP technology employs a thermolabile modification group at the 3′ hydroxyl that blocks dNTP incorporation at low temperatures; when released at higher thermal cycling temperatures, it produces a suitable dNTP substrate for DNA polymerase incorporation (3,4). CleanAmp™ 7-deaza-dGTP is described herein as an effective and simple method that greatly improves and in many cases enables specific amplification of GC-rich regions of varying complexity.

Materials and methods

PCR protocols for the amplification of targets with varying percent GC content using several dNTP combinations (Figure 1) consisted of 1× PCR buffer (supplemented with MgCl₂ to 2.5 mM; Invitrogen), 1.25 U Taq DNA polymerase (Invitrogen), 0.1 µM BRAF primer pair, 0.2 µM all other primer pairs (TriLink BioTechnologies, Inc.), 0.2 mM dNTPs (standard dNTPs, New England Biolabs; CleanAmp™ dNTPs, TriLink BioTechnologies, Inc., Cat. no. N-9501 or N-9505), and 5 ng human genomic DNA as template (Promega). Reactions with 7-deaza-dGTP or CleanAmp™ 7-deaza-dGTP (both from TriLink BioTechnologies, Inc., Cat. no. N-2011 or N-9515, respectively) used a dNTP mixture of the following composition: 0.2 mM d(A,C,T)TP, 0.05 mM dGTP, and 0.15 mM 7-deaza-dGTP. All 25-µL reactions were performed in quadruplicate and run on a Bio-Rad Tetrad 2 thermal cycler with a thermal cycling protocol of 95°C (10 min); [95°C (40 sec), X°C (1 sec), 72°C (1 min)] 35 cycles; 72°C (7 min) where X (annealing temperature) varied depending on the target (ACE 66°C; BRAF 64°C; B4GN4 57°C; GNAQ 66°C). A 1-s annealing time was found to be important for specificity and was used throughout these studies. The GNAQ target was amplified for 40 cycles. After PCR, 20 µL each sample was analyzed by agarose gel electrophoresis. The primer sequences used in these studies were: ACE (156 bp; 60% GC-rich: 5′-GGGATGGTTGCCCAGGCTGGA-3′ and 5′-TGATGGGTGCCGTGGTCTCCT-3′) (1); BRAF (185 bp; 75% GC-rich: 5′-CTCGGTTATAAGATGGCGGCGCTGA-3′and 5′-AGTCGGGAGGGCGGCAGGGT-3′) (1); B4GN4 (719 bp; 78% GC-rich: 5′-GCCTGGAGCCCACCGAG-3′ and 5′-CGTGCGCTGCCAGTCGA-3′) (5), and GNAQ (642 bp; 79% GC-rich: 5′-CCTCCTTCCCCGGGAACAGGC-3′ and 5′-CCGAGGGTGCGGGAGCAGTAG-3′) (1).

Figure 1. PCR amplification of targets with varying percent GC content: (Click to enlarge)

The experimental conditions for Figure 2 were similar to those listed for Figure 1 with the following additions: only CleanAmp™ versions of dNTPs and 7-deaza-dGTP were used (CleanAmp™ 7-deaza-dGTP Mix, TriLink BioTechnologies Inc., Cat. no. N-9504); all primers were used at 0.2-µM concentration; human genomic DNA template was present at four concentrations (0.5, 5, 50, and 500 ng); the annealing temperatures (X°C) for AGT and GNB3 were 61°C and 55°C, respectively, and 35 thermal cycles were performed. The following additional primer sequences were used: AGT (242 bp; 61% GC-rich: 5′-CCTGGAAGAGGTCCCAGCGTGAGT-3′ and 5′-GACAAGACCAGAAGGAGCTGAGGG-3′) (1) and, GNB3 (92 bp; 61% GC-rich: 5′-CTCCCACGAGAGCATCATCTG-3′and5′-GCAGT TGAAGTCGTCGTAGCC-3′) (1).