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Improved PCR Amplification of VNTR Locus D1S80 Using PNA(Peptide Nucleic Acid) OligomersDaniel B. Demers, Elizabeth T. Curry, and Amanda C. Sozer Use of the polymerase chain reaction (PCR) to amplify variable number of tandem repeat (VNTR) loci has been widely used in genetic typing. Unfortunately, preferential amplification of small allelic products relative to large allelic products may result in incorrect or ambiguous typing in a heterozygous sample. The mechanism for preferential amplification has not been elucidated. Recently, PNA oligomers (peptide nucleic acids) have been used to detect single base mutations through PCR clamping. PNA is a DNA mimic that exhibits several unique hybridization characteristics. In this report we present a new application of PNA which exploits its unique properties to provide enhanced amplification. Rather than clamping the PCR, PNA is used to block the template, making it unavailable for interstrand and intrastrand interactions while allowing polymerase to displace the PNA molecules and extend the primer to completion. Preferential amplification is reduced and overall efficiency is enhanced. Use of the polymerase chain reaction (PCR) to amplify variable number of tandem repeat (VNTR) loci has been widely used in gene mapping, linkage studies, diagnostics, forensics, and paternity testing (1-5). The PCR is particularly useful when DNA quality and quantity are limited. However, the PCR is not without its problems. In regards to the amplification of VNTR loci, preferential amplification, anomalous products, and length limitations may be encountered. Preferential amplification refers to the inverse relationship between the size of the VNTR allele and the efficiency of amplification such that small allelic products may be preferentially amplified when present in a heterozygous state with a large allele. In some circumstances, differential amplification may result in dropout of the larger allelic product and misclassification of a heterozygous individual as homozygous for the over-amplified smaller allele. This mechanism has not been elucidated although Walsh et al. (6) suggested that, in some cases, it is the result of incomplete denaturation, differential priming, limiting enzyme, or small sample size. Anomalous PCR products, or ladder-like patterns, are often generated during the PCR of short tandem repeats (STRs). These complex patterns can make genotypic interpretation difficult. It has been proposed that these anomalous DNA fragments are the result of DNA polymerase slippage during primer extension (7-9) and recombinational processes such as 'out of register' annealing of truncated PCR product (10,11) and 'out of register' template switching during DNA synthesis (12-14). Traditionally, the PCR has been limited to target sequences less than 3-4 Kb in length. However, recent work with 'Long PCR' has identified premature termination of primer extension, due either to template nicking, incorporation of mismatched base pairs or premature dissociation of polymerase, as a possible mechanism for the length limitations of the PCR (15-17). Modifications to the PCR buffer and inclusion of a polymerase with 3' exonuclease activity has allowed the amplification of unique target sequences of 10-40 Kb in length. The mechanisms involved in preferential amplification, the production of anomalous products (ladder-like products), and the length limitations for PCR amplification are complex. Usually these phenomena are discussed in isolation without consideration of the others. However, it is feasible that they are interrelated and that the complex milieu of a PCR simultaneously sustains several interstrand and intrastrand interactions. The results of our work suggest that another mechanism may play a role in premature termination: reannealing of complementary template (complete or truncated) and subsequent clamping of the PCR. The objective of our research was to attempt to block the repeat units of a VNTR locus during the PCR amplification, but not prevent the product from being used as template for primer extension. Peptide nucleic acid (PNA) was the molecule selected to accomplish this. Peptide nucleic acid is a DNA mimic in which the deoxyribose-phosphate backbone has been replaced by an oligoamide consisting of N-(2-aminoethyl)glycine units (18,19). PNA mimics DNA in terms of its ability to recognize and bind to complementary nucleic acid sequences but does so with higher thermal stability and specificity than corresponding oligodeoxynucleotides. However, a single base mismatch in a PNA-DNA duplex is much more destabilizing than in the corresponding DNA-DNA duplex. Furthermore, PNA cannot function as a primer for DNA polymerase. The VNTR locus D1S80 was selected as a model to test the system. It consists of a 16 bp repeat with at least 29 alleles ranging in size from 369 to 801 bp corresponding to 14 and 41 repeats, respectively (20,21). The PNA molecules were designed to block the repeat sequence at the D1S80 locus during amplification, but at the same time not prevent the polymerase from displacing them and extending the primers to completion (Fig. 1). To accomplish this the PNA had to anneal to its target sequence at a relatively high temperature and yet be destabilized enough at extension temperatures to be displaced by the polymerase. It would also require a concentration high enough to compete with the build-up of product during later rounds of the PCR but not clamp the PCR as it has been previously shown to do so effectively (22). The composite PNA, synthesized by PerSeptive Biosystems (Framingham, MA) was the following: H-CTT(G/T)CCGGTGGTC(C/T)TC-NH2 The sequence of the primers according to Kasai et al. (20) were the following: 5'-GAAACTGGCCTCCAAACACTCCCCGCCG-3' (forward primer) The forward primer was end-labeled with T4 polynucleotide kinase and [g-32P]ATP. The PCR amplification of DNA samples was performed as previously described (23). The methodology was optimized using purified K562 DNA (Promega Corp., Madison, WI) and tested using DNA from routine paternity testing casework. Sequence analysis of selected D1S80 alleles was performed by direct sequencing of PCR product using a Sequenase Version 2.0 kit (US Biochemicals). Figure 2 shows the result of a PCR experiment using K562 DNA and increasing amounts of PNA. Differential amplification is readily apparent when PNA is absent as evidenced by the less intense upper band (allele with 29 repeats). As the concentration of PNA increases the intensity of the larger fragment increases up to a maximum at ~1.2-1.8mM PNA. Since PNAs are not consumed in the amplification process, the ratio of PNA to template decreases with each cycle as more template is produced. Therefore, the kinetics of PNA annealing are constantly changing in the reactions. To evaluate these changes and determine where in the PCR process PNA exerts its effect, K562 DNA was amplified without PNA and with 0.9, 1.2 and 1.5mM of PNA for 10, 20 and 30 cycles. Results are shown in Figure 3. After 10 cycles of the PCR no detectable product was observed. With 20 cycles of PCR amplification, product can be detected in each lane. No improvement in signal was observed when PNA was present and may have actually reduced the efficiency of amplification, presumably through clamping. However, after 30 rounds of the PCR and a theoretical 109-fold increase in product, enhancement is readily apparent. To evaluate the general application of PNA for enhancing PCR at the D1S80 locus, DNA specimens from routine paternity casework were randomly selected. Each was amplified with (1.5mM) and without PNA. Figure 4 shows the results obtained for nine specimens ranging from 433 bp to 657 bp as determined by comparison to a D1S80 allelic ladder (Perkin-Elmer Cetus, Norwalk, CT). Each DNA specimen demonstrated enhanced amplification, particularly of the larger allele, when PNA was present. It was most apparent in DNA samples demonstrating >6 repeats difference in allele size (samples 2, 4, 5 and 8). However, amplification of close heterozygotes (samples 1, 3 and 6) and apparent homozygotes (samples 7 and 9), also appeared to benefit from the presence of PNA as evidenced by the increased product yield. A total of 85 specimens were tested. Of those, 64 (75%) demonstrated a noticeable improvement in the amplification of one or both alleles. D1S80 alleles refractory to PNA enhancement were presumed to have polymorphisms within the repeat units that were not represented by the composite PNA. Therefore, sequence analysis was performed to better characterize alleles that were both responsive and nonresponsive to PNA enhancement. Sequence analysis of 11 alleles encompassing 133 repeat units revealed additional polymorphic sites within the D1S80 locus. In addition to the G/A and C/A polymorphic sites at positions 3 and 13 respectively, of the repeat, polymorphisms were identified at positions 4 (G/A), 10 (C/A) and 11 (G/A). An additional polymorphism was also identified at position 13 (C/A/G). These polymorphisms were represented in nine different iterations of the repeat. Table 1 gives the sequence of the different repeat units with their frequency of occurrence. To evaluate the compatibility of 'Long PCR' and PNA methodologies, K562 DNA was amplified using standard buffer and modified buffer from the Expand Long Template PCR System (Boehringer Mannheim) without and with PNA (1.5mM). Previous experiments (data not shown) demonstrated reduced amplification efficiency if the Expand kit enzyme mix containing Taq and Pwo DNA polymerases was used, with or without PNA, in the amplification of D1S80. Therefore, the Expand kit buffer was used without the enzyme mix. The modified Expand kit buffer was found to enhance the PCR amplification of D1S80, nearly as much as PNA, but with a concomitant increase in nonspecific high and low molecular weight product (Fig. 5). Enhancement with PNA consistently results in a reduction in the amount of nonspecific product. The use of PNA and the modified buffer together appeared to give more enhancement than either PNA or modified buffer alone. The amount of nonspecific product generated when PNA and modified buffer are used together was intermediary to that obtained with standard buffer without PNA and standard buffer with PNA. We have presented a method using PNA to enhance the efficiency of the PCR amplification of VNTR alleles. If properly designed and present during PCR amplification at the appropriate concentration, PNA allows fragments of different size to be more effectively and more evenly amplified. Differential amplification is less apparent and as a result, the risk of misclassification is greatly reduced. In addition, fragments of the same or similar size are also amplified more effectively when PNA is included in the PCR. Peptide nucleic acid enhancement of the PCR process appears to occur during later rounds of amplification. Reduced amplification efficiency after 20 cycles in the presence of PNA suggests that excess PNA molecules are initially, to some extent, clamping the PCR. This suggests that the accumulation of product in the later rounds of the PCR may be the cause, at least in part, for preferential amplification of VNTR loci. It is in later cycles that the PCR components are depleted while product is greatly increased. Complementary strands of the template may be reannealing before extension takes place thereby blocking primer extension and possibly primer binding. Since larger alleles contain more repeat units, they anneal at a faster rate, thereby reducing the efficiency of the PCR amplification of those fragments. Blocking of the repeat units by PNA molecules prevents reannealing of complementary template strands, allowing primer extension to occur without impediment. The degree of enhancement obtained when randomly selected samples were amplified with PNA was variable. It was believed that such variation reflected sequence and length variation present within the D1S80 repeat. Sequence analysis of 11 D1S80 alleles did indeed reveal additional polymorphisms not represented by the composite PNA molecules. Thus, blocking of those alleles may have been incomplete, resulting in reduced enhancement. Redesign of the PNA composite to include those polymorphisms substantially represented by D1S80 alleles, and in proportion to their relative frequencies should further improve the PNA method. Long PCR methodology utilizing a modified buffer without the enzyme mixture provided enhanced PCR amplification of D1S80 alleles, but with a concomitant increase in nonspecific product. Further enhancement is obtained by including PNA in the PCR, and nonspecific product is reduced. Inclusion of PNA in the PCR, regardless of other conditions, appears to consistently reduce the generation of high and low molecular weight nonspecific products. We are grateful to Michael Egholm, PerSeptive Biosystems, Framingham, MA, for providing the PNA molecules used in this research.
Table 1. Repeat sequences observed at VNTR locus D1S80.
Table 1. A total of 133 repeats were sequences from 11 different alleles. The first two repeats of each allele invariably consisted of the truncated repeat AGCCCAAGGAAG followed by ACAGACCACAGGCAAG. The remaining 121 repeats consisted of the nine sequences listed. The frequency (number out of 121) of each repeat sequence is given to the right. -, same base as first sequence listed, polymorphic sites at position 3 and 13 of the repeat identified by Kasai et al. (20). Figure 1. Schematic diagram of the modified PCR reaction in the presence of PNA.
Following template denaturation, PNA molecules anneal to and block the DNA template. A separate PNA annealing step (at about 78°C) may be used, or PNA annealing may occur while cooling to the primer annealing temperature. Once DNA primer molecules have annealed to the template, then the temperature is raised for primer extension. During primer extension, PNA molecules are displaced from the template allowing primer extension to proceed to completion. Figure 2. Optimization of the PNA concentration required for enhanced amplification of K562 DNA.
K562 DNA was amplified without (lane 1) and with increasing amounts of PNA (lanes 2-9). The allele size (in repeats) is given in the left. Primer extension was carried out at 72°C. Figure 3. A comparison of PNA enhancement of the PCR at 20 and 30 cycles of amplification.
K562 DNA was amplified with PNA (0.9, 1.2, and 1.5 mM) and without PNA for 10 cycles (data not shown), 20 cycles, and 30 cycles. Primer extension was carried out at 76°C.
Nine randomly selected DNA samples (samples 1-9) and K562 DNA (sample 10) were amplified by the PCR with PNA (1.5 mM) and without PNA. Primer extension was carried out at 76°C. Allele size ranged from 18 to 32 repeats. -, no PNA added; +, PNA added. Figure 5. A comparison of the efficiency of the PCR of K562 DNA at VNTR locus D1S80
using 1) standard conditions, 2) PNA, 3) modified buffer from Expand Long Template PCR System, and 4) both PNA and modified buffer. High and low molecular weight nonspecific product is shown above and below the D1S80 alleles respectively, -, component absent, +, component present. Go to proceedings home page
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