Primers that amplify a fixed-length product from telomeric tandem hexamer repeats
Relative average telomere length can be measured by QPCR using primers that hybridize the telomeric hexamer repeats, because the number of binding sites for the primers increases as average telomere length increases. Our original tel1 and tel2 primers for telomere length measurement by singleplex QPCR (1
) are both able to prime at multiple locations along the tandem repeats of telomeric DNA. They therefore generate a series of products of various sizes, some of which melt at temperatures high enough to overlap the melting curve of the scg's amplicon. Consequently, ‘clean’ reads, at a high temperature, of the SYBR Green I fluorescence signal from the scg's double-stranded amplicon, without any interfering signal from double-stranded telomere PCR products, as required for successful MMQPCR, are not possible when tel1 and tel2 are the telomere primers.
To solve this problem, we designed a pair of telomere primers, telg, ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT and telc, TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA, that generate a short, fixed-length product (). Only telg is able to prime DNA synthesis along native telomeric DNA sequences. The telc primer is blocked from priming native telomeric DNA by a mismatched base at its 3′ terminus. However, telc is able to hybridize along various stretches of the telg primer extension product, and exactly one configuration of those hybridizations allows the priming of DNA synthesis, thereby enabling the generation of a single, fixed-length product. This is achieved by introducing a nucleotide change in telg at the third base from the 3′ end, such that the last three bases of the telg and telc primers overlap with perfect complementarity. This overlap is not sufficient to allow the native telg and telc primers to prime each other efficiently, so primer dimer formation is undetectable over the range of cycles that telomere length quantitation occurs. However, when the telg extension product is hybridized to telc, this three base overlap is the only site where the 3′ end of telc can efficiently prime DNA synthesis. The resulting PCR product is, therefore, of fixed length, and three bases shorter than the sum of the lengths of the two primers used to generate it. The sharp melting curve for this product (green curve in ) is consistent with specific, fixed-length product formation, and agarose gel electrophoresis in 6% gels revealed only the expected 79 bp product (data not shown). also demonstrates that the melting curve for the telomere PCR product is well separated from the melting curve for the albumin PCR product (blue curve in ), allowing the SYBR Green I signal from albumin to be read at a temperature that fully melts the telomere PCR product.
Figure 1. In Cycle 1 the telg primer hybridizes to native telomere sequences and primes DNA synthesis. The telc primer hybridizes native telomere sequences but cannot prime DNA synthesis, due to its 3′ terminal mismatch. When hybridized to each other as (more ...)
Figure 2. Melting curves following 25 cycles of amplification (thermal profile given in Materials and methods section) of 150 ng of human genomic DNA with telomere primers only (green curve), albumin primers only (blue curve) or both primer sets (orange curve). (more ...)
Primer design for single copy genes (albumin and beta-globin)
Primers were designed so that the scg amplicon would melt at a much higher temperature than the telomere amplicon. Fluorescent signal from the scg amplicon could then be acquired at a temperature high enough to completely melt the telomere amplicon, eliminating its contribution to the signal, but low enough to keep the scg amplicon double-stranded and therefore able to bind SYBR Green I.
The primers for amplification of the scg albumin are albu: CGGCGGCGGGCGGCGCGGGCTGGGCGGaaatgctgcacagaatccttg and albd: GCCCGGCCCGCCGCGCCCGTCCCGCCGgaaaagcatggtcgcctgtt. The predicted product size is 98 bp. The primers for amplification of the scg beta-globin are hbgu: CGGCGGCGGGCGGCGCGGGCTGGGCGGcttcatccacgttcaccttg and hbgd: GCCCGGCCCGCCGCGCCCGTCCCGCCGgaggagaagtctgccgtt. The predicted product size is 106 bp. Capitalized bases are non-templated 5′ tag sequences that confer a very high melting temperature on the resulting PCR product. Please note that the 5′ tag sequences for the albumin primers are identical to those used in the beta-globin primers. Note also that the two GC-rich 5′ tagging sequences in each primer set are very different from each other; if they were the same, hairpin formation shutting down amplification would be likely to occur during the PCR.
The addition of a GC-clamp to the 5′ end of a PCR primer to raise the melting temperature of one end of the PCR product is common practice when screening a gene for point mutations by Denaturing Gradient Gel Electrophoresis (2
). We reasoned that by attaching 5′ GC-clamps to both of the primers used to amplify the scg, and keeping the targeted genomic sequence short, we would be able to generate a PCR product with a very high melting temperature. shows that the Tm
for the doubly GC-clamped albumin PCR product is above 91°C. Agarose gel electrophoresis in 6% gels revealed only the expected size product. Similar results were obtained for the doubly GC-clamped beta-globin PCR product (data not shown).
The 5′ GC-clamps, also ensure that both of the primers used to amplify the scg have Tm
values for their amplicon that are higher than the Tm
of the telomere PCR product. The benefits of this design are discussed below (see Thermal profile and cycling design section). An analysis using the OligoAnalyzer program (www.idtdna.com
) indicated that all four scg primers (albu, albd, hbgu and hbgd) have Tm
values greater than 84°C in the buffer composition used in this study.
Thermal profile and cycling design
In Stage 1 of the thermal cycling protocol, the AmpliTaq Gold DNA polymerase is heat-activated, and the genomic DNA sample is denatured. In Stage 2, two cycles of relatively low temperature are needed to effectively anneal and extend the telomere primers, due to the presence in those primers of purposely introduced mutations that prevent formation and amplification of primer dimer PCR products (1
In Stage 3 the repeating cycle begins with a denaturation, an annealing and an extension step with signal acquisition that are typical of conventional QPCR. These are followed by two unconventional steps: an incubation at 84°C for 10 s and an incubation at 88°C for 15 s with a second signal acquisition. Heating to 84°C melts the early-amplifying telomere product, releasing DNA polymerase (which binds double-stranded, but not single-stranded DNA; 3) for work on the scg PCR product, where DNA synthesis can proceed, due to the high annealing temperatures (above 84°C) of the scg primers, and the ability of Taq DNA polymerase to maintain robust activity even at 84°C (4
In conventional multiplex PCR, high concentrations of the earliest amplifying product often inhibit subsequent amplification of less abundant templates, due to the above-mentioned binding up of DNA polymerase by the early product. The usual recommended solution is to limit the primer concentrations for the more abundant target sequence, so that less product is formed, leaving enough DNA polymerase unbound and free to continue copying the less abundant template. But lowering primer concentrations often results in a reduced PCR efficiency, or even a complete failure to amplify the target sequence. Reduced efficiencies also contribute to greater variation in Ct values between replicates. The 84°C incubation step in MMQPCR eliminates the need to limit the primer concentrations for the more abundant template, releasing polymerase from even high concentrations of the corresponding PCR product, so that the second product can be synthesized efficiently.
Heating further to 88°C for the second signal acquisition step ensures that the telomere PCR product is completely melted and unable to interfere with the collection of the rising SYBR Green I fluorescence signal from the accumulating scg amplicon.
Validity of the MMQPCR method over the natural range of telomere lengths
shows amplification curves collected at two different temperatures (74°C and 88°C) for three reference human genomic DNA samples previously shown to have high, middle or low average telomere lengths (approximately a 3-fold range of telomere lengths). Based on the melting profiles presented in , the 74°C reads should detect both telomere and albumin PCR products, and the 88°C reads should detect only the albumin product. However, because the albumin template is much lower in copy number than the telomere template in each DNA sample, the 74°C Ct values, all collected when the corresponding albumin signals were still at baseline, are measures of telomere amplification only. (We have confirmed, in reactions without the telomere primers, that the single copy gene signal rises above baseline at essentially the same cycle number whether collected at 74°C or 88°C.) Even the sample with the shortest telomeres (~1670 bp), and therefore the most right-shifted amplification curve (blue curve), crosses threshold at a cycle number when the albumin gene's amplification signal is still at baseline. In the present study of 95 whole blood DNA samples from subjects aged 5–94 years, each sample's scg amplication signal was at baseline when the Ct for the corresponding telomere signal was collected.
Figure 3. MMQPCR of 20 ng of each of three reference human DNA samples previously shown to have long telomeres (orange curves), middle-length telomeres (green curves) or short telomeres (blue curves). No template control amplification curves are in black. Top panel: (more ...)
Independent standard curves for telomere and single copy gene
shows two independent standard curves, one for the telomere repeats and another for the scg albumin, determined for the Standard DNA by acquiring the SYBR Green I fluorescence signal at two different temperatures (74°C for the telomere signal and 88°C for the albumin signal) in each cycle of Stage 3 of the cycling protocol. This same DNA sample was used to generate two standard curves for each separate PCR reaction plate in this study. In this semi-log plot of DNA concentration versus cycle threshold, both curves are linear over the 81-fold DNA concentration range. The PCR efficiencies for both telomere and albumin amplifications were greater than 90% and approximately equal. For this particular Standard DNA sample, at each DNA concentration the Ct for albumin occurred approximately six cycles later in cycling than the Ct for the telomere repeats.
Figure 4. Standard curves used to determine relative T/S ratios. Five concentrations of a standard human genomic DNA sample spanning an 81-fold range were prepared by 3-fold serial dilutions (150 ng, 50 ng, 16.7 ng, 5.55 ng and 1.85 ng per well), and aliquoted (more ...)
Correlation between mean TRF lengths and relative T/S ratios
To test the validity of the MMQPCR approach to telomere length measurement, we compared the relative telomere lengths (average T/S ratios) in whole blood DNA samples from 95 individuals, aged 5–94 years, measured in triplicate by MMQPCR, to the mean TRF lengths of these same DNA samples as measured by the traditional Southern blot approach (1
). shows the strong correlation in relative telomere lengths measured by these very different techniques (R2
= 0.844). This correlation is higher than the correlation we reported previously (1
) for T/S ratios measured in these same samples by singleplex QPCR versus their mean TRF lengths (R2
Figure 5. Correlation of relative T/S ratios measured by MMQPCR with albumin as the single copy gene, and mean TRF lengths determined by Southern blot analysis, in whole blood DNA samples from 95 individuals. Each T/S value is the average of triplicate measurements; (more ...)
Reproducibility of T/S ratio measurements
To examine the intra-assay reproducibility of T/S measurements by MMQPCR, we determined the coefficient of variation (standard deviation divided by the mean) for T/S for each of the 95 DNA samples assayed in triplicate in a single run of the MMQPCR assay, using albumin as the scg. The intra-assay geometric mean of the coefficient of variation was 5.22%. To examine inter-assay reproducibility, we repeated the measurements of T/S in the same 95 DNA samples, again in triplicate, on another day, taking care that the specific MyiQ PCR machine and reaction well positions occupied by each DNA sample were different in these two independent runs of the assay. shows the strong correlation between the average T/S ratios determined by the first and second runs (R2 = 0.91). The slope of the linear regression line through the data was near unity, and the y-intercept near zero, as expected. The coefficient of variation for each of the 95 pairs of average T/S values from the two independent runs was determined. The inter-assay geometric mean of the coefficient of variation was 3.13%.
Figure 6. Reproducibility of relative T/S ratios in independent runs of the MMQPCR assay. The same 95 DNA samples assayed in were assayed again the next day, taking care that the specific MyiQ PCR machine and reaction well positions occupied by each DNA (more ...)
T/S ratios are independent of the single copy gene used
To test whether using beta-globin, instead of albumin, as the scg might alter apparent relative telomere lengths, we repeated the measurements of T/S in the same 95 DNA samples, in triplicate, in two separate runs, substituting the beta-globin primers for the albumin primers. plots the average T/S values from the two runs with albumin as the scg (x-axis) versus the average T/S values from the two runs with beta-globin as the scg (y-axis). The T/S values obtained with albumin correlated highly with those obtained using beta-globin (R2 = 0.934).
Figure 7. Correlation between T/S ratios obtained with albumin as the single copy gene versus beta-globin as the single copy gene. Relative T/S ratios were measured in the same 95 DNA samples, in triplicate, in two separate runs, substituting the beta-globin primers (more ...)