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The Effect of Single Mismatches on Primer Extension.

Allele-specific PCR (AS-PCR) [2] is commonly used to discriminate between alleles that have single-nucleotide variants (SNVs) and well-defined insertions and deletions (1, 2). The 3' end of one primer is designed to hybridize to the variant base and selectively amplify the allele of interest. When the target base opposite the primer's 3' nucleotide is complementary to the primer, the allele is amplified efficiently, but when there is a mismatch, amplification is inhibited. Mechanistic descriptions of how mismatches reduce the ability of DNA polymerase to incorporate nucleotides depending on mismatch type and position have been proposed (3, 4).

Studies of mismatch extension rates fall into 2 categories. Model systems unrelated to PCR use radioactive or fluorescently labeled substrates that are quantified to calculate relative specificity or extension rates from mismatched primer-template pairs (5-8). However, buffer systems and temperatures are rarely applicable to AS-PCR. Alternatively, PCR can be used to judge the effects of mismatches on polymerase activity. Multiple investigations have shown reduced PCR yield or increased quantification cycles ([C.sub.q]) (9-14). However, for endpoint assays it is difficult to know when to stop cycling, as too few cycles may not reveal any product and too many cycles may attenuate any differences because of the plateau phase of PCR. This can be solved by real-time analysis, but there is no guarantee that the assay is optimized or that the PCR results are attributable to primer extension alone. Other reactions in PCR such as denaturation and annealing can confound conclusions about primer extension derived from PCR.

To describe primer extension in absolute quantitative units, extension of a hairpin was monitored with a double-stranded DNA (dsDNA) dye on a stopped-flow instrument. Highly stable self-priming templates minimized the impact of annealing kinetics and thermodynamic stability on measured extension rates. Unlike prior model systems, buffers, dyes, and temperatures commonly used in rapid cycle PCR (15) were employed, and a deletion mutant of Thermus aquaticus (Taq) polymerase with improved specificity was used (16). Using a dsDNA dye commonly found in quantitative PCR is a convenient method to monitor product accumulation and report reaction velocities in units of nucleotides per second per polymerase (nt/s/poly).

Materials and Methods


Oligonucleotides were synthesized and then purified via high-pressure liquid chromatography (Integrated DNA Technologies). A total of 132 mismatched template sequences were derived from 3 "matched" (perfectly complementary) hairpins. Only single-base mismatches were designed. For reactions that did not reach completion within a 30-min time frame, additional oligonucleotides were synthesized as fluorescence standards corresponding to fully extended hairpins that were purified by polyacrylamide gel electrophoresis. All oligonucleotides were quantified by absorbance at 260 nm after being fully denatured in 1 mol/L NaOH (17) and had predicted melting temperatures ([T.sub.m]s) above 82[degrees]C with [Mg2+] = 3 mmol/L and [Na+] = 20 mmol/L at 65[degrees]C (18). Matched templates were melted on an HR-1 (BioFire) with a 0.3[degrees]C/s ramp rate and had melting temperatures >87[degrees]C under assay conditions. Oligonucleotide sequences, [T.sub.m]s, and predicted Gibbs energy at 65[degrees]C are shown in Fig. 1 and Table 1 in the online Data Supplement (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www. Oligonucleotide hairpins were designed to have high stem [T.sub.m]s, approximately 50% GC content in the extendable region, and limited additional secondary structure. The 3 matched hairpins had identical loop sequences with differing lengths and sequences for the stem and overhang regions.


KlenTaq 1 (DNA Polymerase Technology) and Taq (New England BioLabs) were used in this study. KlenTaq performs better at 3' mismatch discrimination (19) and extends quickly (20) compared to most DNA polymerases under PCR conditions. Quantification was done on SDS polyacrylamide gels (Mini-PROTEAN TGX Stain-Free Precast Gels, Bio-Rad) with a 5 mmol/L Tris-HCl, 192 mmol/L glycine, 0.1% SDS, and pH 8.3 running buffer. Quantification was performed relative to a KlenTaq standard that was previously quantified via absorbance at 280 nm on a Nanodrop 2000 (ThermoFisher) using an absorptivity of 6.91 x [10.sup.4] [M.sup.-1] [cm.sup.-1] (16, 20).


Nucleotide incorporation was investigated with a previously described polymerase activity assay (20). The assay was performed with a stopped-flow instrument (BioLogic). Reactions were initiated by mixing dNTPs with the solution containing polymerase and oligonucleotide at an output flow rate of 11 mL/s. The estimated dead time for the reactions was 5.4 ms. Reactions were performed at 65[degrees]C unless otherwise noted. Polymerase stocks were diluted in 50 mmol/L Tris pH 8.3, 500 jug/mL BSA, and 0.03% Tween-20. Reaction conditions were 50 mmol/L Tris pH 8.3, 500 jug/mL BSA, 3 mmol/L MgCL, 200 jumol/L each dNTP (Bioline), 1x EvaGreen dsDNA dye (Biotium), 400 nmol/L template, and 20 nmol/L polymerase. For the Taq and KlenTaq activity comparison, the reactions included 0-50 mmol/L KC1.

For position studies, 8 mismatched base pairs were examined at 0, 1, 2, 4, 6, 8, and 10 bases from the 3' end with use of 112 templates derived from matched templates 1 and 2 (Fig. 1, and see Table 1 in the online Data Supplement). Single mismatches from "symmetric pairs" [e.g., A * C and C * A mismatches (primer * template)] were used as previous work indicated symmetric mismatches were similar in effect (11-13)?

All 3' mismatches (position 0) were studied with use of 36 mismatched templates derived from all matched template sequences (Fig. 1, and see Table 1 in the online Data Supplement). For the temperature study, 9 oligonucleotides were investigated in the range of 40-75[degrees]C. Taq and KlenTaq activity was compared by use of 3' mismatches derived from template 3. For the Michaelis-Menten study, 7 oligonucleotides, including matched templates and templates with G * T and T * T mismatches, were used at concentrations between 10 and 1200 nmol/L.


Reactions that reached exhaustion within 30 min were calibrated by normalizing the minimum fluorescent value of each progress-curve to zero, and the maximum fluorescent value to the average number of extendable bases per polymerase. Then, the initial slope of the curve corresponds to the average initial rate in nucleotides per second polymerase (nt/s/poly). Reactions that did not reach exhaustion within 30 min were calibrated by use of negative controls (all components except polymerase) and positive controls (all components with synthesized standards of fully extended templates) before being normalized between zero and template exhaustion (20). Results reported are the mean initial rate and standard deviation of several "shots" by stopped-flow, given in nucleotides per second per polymerase (nt/s/poly).

To determine sample and shot variability, 10 experiments consisting of 6 stopped-flow shots each were run with the matched template 1. The average CV between shots was 6.64%, while the average CV between samples was 7.58%. The assay's detection limit was determined by recording 6 replicate negative controls (all reaction components besides polymerase) of the matched template 1 for 30 min. Then, the negative controls were calibrated via positive controls as described above before an initial slope measurement was taken. The assay detection limit (mean + 2 SD) was 0.004 nt/s/poly.

For the Michaelis--Menten parameters, initial rate vs concentration data was fit by nonlinear regression (21) to the single-substrate Michaelis--Menten equation (22) to obtain [K.sub.m] and [] values for each matched and mismatched template.

Differences between mismatch rates were identified by ANOVA followed by multiple t tests with Sidak correction and [alpha] at 0.050 (23).



The matched "template 1" had an extension rate of 205 [+ or -] 11 nt/s/poly, while rates for mismatches decreased as each mismatch type approached the 3' end (Fig. 2A, and see Table 2 in the online Data Supplement). At 10 bases, mismatched templates had rates between 190 and 208 nt/s/poly, comparable to that of the match. Inhibition by mismatches at positions 10 and 8 base pairs from the 3' end were <7% and 31%, respectively. Mismatches 6 bases from the 3' end measured between 169 [+ or -] 15 for the G * A mismatch (primer * template) and 33.0 [+ or -] 2.2 nt/s/poly for the C * C mismatch, while rates for mismatches 4 bases from the 3' end were similar (156 [+ or -] 10 and 35.9 [+ or -]1.6 nt/s/poly for the G * G and C * C mismatches, respectively). Mismatches one base away from the 3' end were between 14.8 [+ or -] 1.2 (C * A mismatch) and 0.155 [+ or -] 0.005 nt/s/poly (G * A). Mismatches at various positions of template 2 (Fig. 2B, and see Table 2 in the online Data Supplement) showed a similar relationship between rate and proximity of the mismatch to the 3' end of the oligonucleotide.

Extension rates of mismatches from template 1 and 2 (see Fig. 1A in the online Data Supplement) often correlated (positions 1-6; see Fig. IB in the online Data Supplement). There was a negative correlation between extension rates and calculated Gibbs energy (positions 2-8; see Fig. 2A in the online Data Supplement), but not for 3' end or penultimate mismatches (see Fig. 2B in the online Data Supplement).

Inhibition was strongly associated with mismatch type, with C * C, A * A, and G * A mismatched hairpins among the most inhibitory and G * T and C * A mismatches among the least inhibitory at every position across the template sequences (Fig. 2, A and B). Rates varied 2 orders of magnitude between different mismatch types at the same position. For example, 0.155 [+ or -] 0.005 to 13.1 [+ or -] 0.4 nt/s/poly for the G * A and G * T mismatches, respectively, one base from the 3' end for template 1. Relative rates of extension between different mismatch types were not always consistent across all mismatch positions. For example, C * C mismatches were the most inhibitory at positions 4 and 6 from the 3' end, but not at other positions. However, relative inhibition of mismatches was similar between templates 1 (Fig. 2A) and 2 (Fig. 2B) at most positions.


All 12 possible 3r mismatches (position 0) were studied with mismatches derived from all 3 hairpin oligonucleotides at 63[degrees]C (Fig. 3, and see Table 2 in the online Data Supplement). Rates for the matched templates were between 177 [+ or -] 8 and 205 [+ or -]11 nt/s/poly. All 3' mismatches reduced nucleotide incorporation rates by 4020000-fold. Extension rates ranged from 4.55 [+ or -] 0.21 for the template 2 T * G mismatch to 0.008 [+ or -] 0.005 nt/s/ poly for the template 2 G * A mismatch. When comparing extension rates of a single mismatch type on the different templates, the rates varied a maximum of 6.6-fold (T * C mismatch between templates 1 and 3). The relative inhibition of 3' mismatches was similar across the 3 template sequences (Pearson's correlation coefficient r = 0.83, 0.78, 0.91, for templates 1 and 2, 2 and 3, and 1 and 3, respectively).

Fig. 3 in the online Data Supplement shows the average and standard deviation of the mean values for 3' mismatches of templates 1-3 (n = 3). Due to low assay variation between shots and runs (CVs = 6.64%, 7.58%, respectively) the large error bars indicate the effects of sequence variation between templates. Sequence context strongly influences the ranking of less inhibitory mismatches (T * G, G * T, C * T, T * C, A * C, C * A) resulting in similarly large ranges for extension rates. The extension rates of more inhibitory mismatches (C * C, A * A, A * G, G * A) display less variation with changes in sequence context. Ordered from highest to lowest, the mean rate of y mismatches was G * T > T * G > C * T > C * A > A * C > T * C > T * T > G * G > C * C > A * A > A * G > G * A, with significant differences between the fastest (G * T, T * G, C * T, C * A) and slowest (T * T, G * G, C * C, A * A, A * G, G * A) mismatches.


Extension rates were observed within a temperature range of 40-75[degrees]C. All matched templates had optimum extension rates between 68[degrees]C and 70[degrees]C (Fig. 4A). The optimum temperature for 3' -mismatched templates was between 55[degrees]C and 60[degrees]C (Fig. 4B). At 40[degrees]C, extension rates for the matched templates were 8%--12% of the maximum rates, while extension rates for mismatched templates were between 15% and 40% of their maxima. At 75[degrees]C the matched and mismatched templates were between 62% and 79%, and 12% and 37% of their maximum extension rates, respectively. Fig. 4C shows the rate of extension for different 3'-mismatched sequences as a percentage of the rate of the matching sequence as a function of temperature.


KlenTaq and Taq extension rates were investigated with respect to KC1. Addition of 50 mmol/L potassium chloride to the reaction increased extension rates of matched templates by Taq by 8.6-fold and decreased rates from KlenTaq by 63% (see Fig. 4A in the online Data Supplement). Absolute extension rates from 3' mismatches and discrimination relative to matched templates decreased with increasing KC1 for both enzymes (Fig. 4, B and C). At 50 mmol/L KC1, discrimination was approximately 6-fold better with Taq than with KlenTaq, a finding that held for G * T, C * T, T * C, C * A, A * C, T * T, and G * G mismatches (data not shown).


The experimental data and nonlinear fit for a representative experiment are shown in Fig. 5 in the online Data Supplement. [] values varied between matched templates (226-335 [s.sup.-1]) and mismatched templates (0.558-8.37 [s.sup.-1]), while apparent Km values varied less (60.3-162 nmol/L and 141-348 nmol/L for matched and mismatched templates, respectively). Specificity constants (22) (k for matched and mismatched hairpins varied 100-1000-fold (Table 1). In all instances, mismatched sequences had greater Km and smaller [] values than the matched sequences from which they were derived.


AS-PCR is used for SNV genotyping and to selectively amplify alleles. Only standard PCR primers are needed, and 2 reactions with 3 primers are used to determine the 3 genotypes of simple SNVs. Analysis can be performed on gels or real-time PCR. Some studies recommend designing AS-PCR primers with an additional mismatch common to both alleles near the 3' end to increase specificity (24). However, this technique limits PCR speed, as an additional 3' antepenultimate mismatch decreases extension of the target allele by 57%--99% (Fig. 1). Additional variations exist, including use of tailed primers and melting analysis so that only 1 reaction is needed (25)? Knowledge of how extension rates vary as a function of position, mismatch type, [KC1], and temperature under PCR conditions can empower the best choice of which position to mismatch, what mismatch to employ, and what temperature and buffer conditions to use. Since there are different classes of SNVs with different mismatch requirements, AS-PCR may be optimal for some but not for others, in which case alternative methods can be considered.

In agreement with previous studies (12, 14, 26), extension rates for all mismatch types decreased as the position of the mismatch approached the 3' end of the primer. Mismatches inhibited extension as far as 6 nucleotides from the 3' end and reduced rates by as much as 2 orders of magnitude when placed 2 nucleotides from the 3' end. Some 3'-penultimate mismatches (e.g., G * A) inhibited extension more than some 3' mismatches (e.g., T * G, A * C, and T * C) (14).

Similar to a prior report (26), extension rates of mismatches 4-8 bases from the 3' end correlated with the predicted thermodynamic stability (18) of the mismatched hairpin (see Fig. 2 in the online Data Supplement). This suggests extension rates of these mismatches are affected by changes in primer--template thermodynamic stability rather than direct mismatch inhibition of polymerase extension. There was little correlation between thermodynamic stability and mismatch inhibition for hairpins with mismatches 0-2 bases from the 3' end, where direct steric changes are expected to alter DNA polymerase-duplex interactions and inhibit activity.

Consistent with prior reports using model systems (5-7, 26, 27) or PCR observations (9, 10, 12-14), 3' mismatches reduced polymerase nucleotide incorporation rates. The amount of inhibition depended on the type of mismatch. Extension rates from purine * pyrimidine mismatches are generally the highest, while purine * purine mismatches are generally slowest, with pyrimidine * pyrimidine mismatches at intermediate rates. The only exceptions are the G * G and C * C mismatches, which are inverted in order of expected rates, and the C * T mismatch with faster extension than typical pyrimidine * pyrimidine mismatches. Most 3' purine * purine mismatches are 2 orders of magnitude slower than pyrimidine * purine/purine * pyrimidine mismatches. Although the extension rates of some mismatches are more than 10 000 times slower than their matched templates, others are <100 times slower, reflecting lower discrimination from their matches.

An alternative method for SNV genotyping is melting analysis of small amplicons (28). Both AS-PCR and melting analysis are simple, inexpensive methods that only require standard PCR primers for genotyping and inexpensive generic dyes for detection. The same 4 classes of biallelic single nucleotide substitutions that are useful in melting analysis (28) are useful in AS-PCR. Variants within each class result in the same heteroduplexes for melting analysis and the same possible 3' mismatches in AS-PCR (Table 2). Class 1 variants require mismatches with relatively high extension rates (on average, 1.2% of the matched templates), making melting a more robust choice for this class because there are large differences in homozygote melting temperatures ([DELTA][T.sub.m]) of 0.8-1.4[degrees]C (28). Strongly discriminatory A * G mismatches are available for class 2 variants that also have large [DELTA] [T.sub.m]s, optimal conditions for both methods. For class 3 and 4 variants, melting is not a good choice because [DELTA]A[T.sub.m]s are <0.4[degrees]C, challenging even high-resolution melting instruments. Instead, AS-PCR is a better choice with discriminatory C * C and A * A mismatches. Class 1 variants make up the majority of human SNVs (66%). They are the hardest to genotype by AS-PCR, but easy to genotype by melting. Conversely, class 3 and 4 SNVs are hard to genotype by melting, but relatively easy to genotype by AS-PCR. Class 2 SNVs are easy to detect by either method. The SNV type determines the best method for analysis.

The optimum extension temperature for 3'-mismatched templates (55-60[degrees]C) is lower than for matched templates (70[degrees]C). The increase in extension rates of 3;-mismatched hairpins at lower temperatures may result from improved binding affinity of the polymerase, as the binding of Taq and KlenTaq polymerases to primer--template complexes is thermodynamically optimal between 40[degrees]C and 50[degrees]C (29). Relative extension rates (mismatched to matched hairpins) typically increase an order of magnitude between 75[degrees]C and 40[degrees]C (Fig. 4C). This suggests that specificity can be improved in AS-PCR by increasing annealing temperatures, using two-step protocols, and limiting cycling times. For example, a 2-step PCR from 90-95[degrees]C to 70-75[degrees] should result in better allele-specific amplification than a protocol with a lower annealing temperature. Nonspecific amplification during preparation is a concern for any PCR assay. The rate of extension of mismatched templates relative to matched templates increases as temperatures decrease. Thus, the use of hot-start methods (30-32) and preparation of PCR at low temperatures (e.g., on ice) coupled with a fast initial ramp to denaturation should improve specificity by limiting the amount of unintended extension that occurs during assay preparation.

Addition of KC1 always inhibits mismatch extension and improves discrimination relative to a matched template, but it has different effects on extension from matched templates for Taq and KlenTaq (see Fig. 4 in the online Data Supplement). With identical buffer conditions, KlenTaq had equivalent or improved discrimination compared to Taq, except at 50 mmol/L KC1. KlenTaq always has higher activity than Taq and is preferred for use in rapid AS-PCR protocols. When improved discrimination is necessary, Taq with >50 mmol/L KC1 could be explored.

Although effects of position, mismatch type, [KC1], and temperature can be predicted, subtle sequence variations also influence extension rates. For example, 3'-mismatched primers with a penultimate G or T are reported to have lower ACqs than those for a penultimate C or A (14). In the current study, when template 2 mismatches (penultimate G) are compared to template 1 and 3 (penultimate C) mismatches, this relationship is not observed. Only the G * G mismatch in template 2 has a lower rate than other templates. However, extension rates from 3'-mismatched template 1 and 3 correlate better (r= 0.91, n = 12) than either template with template 2 (r = 0.85, 0.78; n = 12), suggesting that the ability of DNA polymerase to extend 3' mismatches is dependent on sequence context, but perhaps in a more complicated manner than previously described.

[K.sub.m] values for matched templates are comparable to those reported for DNA polymerases under similar conditions (33)- In the current study, 3' mismatches decreased [] values notably--with the catalytic rate of incorporating a T * T mismatch nearly 3 orders of magnitude less than the rate for a match (Table 1). [K.sub.m] values for 3' mismatches are greater than those for matched templates, suggesting that the 3' mismatches disfavor polymerase binding relative to correctly paired 3' termini. The Km of the template 1 match and the template 2 G * T mismatch are comparable (162 vs 176 nmol/L), suggesting that the sequence context near the 3' end may play an important role in determining polymerase-substrate affinity. The differences in both [] and [K.sub.m] between matched and mismatched templates indicates that mismatches alter polymerase-substrate interactions in multiple ways--3' mismatches directly destabilize polymerase--primer--template binding and reduce the rate of substrate catalysis.

Measured extension rates are a combination of binding, chemical catalysis, DNA translocation, and product release steps. Observed rates are a composite of sequential incorporation events wherein the distance of a base pair from the 3' end increases one base pair with each additional nucleotide incorporation event. Extension rates of mismatched templates vary significantly with mismatch type, position, KC1 concentration, and temperature, but in predictable ways across different primer--template pairs. These results, which show the influence of single mismatches on extension rates under common PCR assay conditions, but without the confounding effects of PCR, can predict and optimize the performance of AS-PCR and can be used to assess the impact of primer--template mismatches during primer design.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: C.T. Wittwer., Clinical Chemistry, AACC.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: C.T. Wittwer, Bio Fire Diagnostics.

Expert Testimony: None declared.

Patents: C.T. Wittwer, 7,387,887.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or final approval of manuscript.


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Nick A. Rejali, [1] Endi Moric, [1] and Carl T. Wittwer [1] *

[1] Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT.

* Address correspondence to this author at: Department of Pathology, University of Utah Medical School, 50 N Medical Drive, Salt Lake City, Utah 84132. Fax +801-581-6001; e-mail

Received September 22, 2017; accepted January 26, 2018.

Previously published online at DOI: 10.1373/clinchem.2017.282285

[2] Nonstandard abbreviations: AS-PCR, allele-specific PCR; SNV, single-nucleotide variant; dsDNA, double-stranded DNA; Taq, Thermus aquaticus; [C.sub.q], quantification cycle; Tm, melting temperature.

Caption: Fig. 1. Matching template sequences, measured [T.sub.m]s, and predicted [DELTA]G values at 65[degrees]C.

Capitalized nucleotides indicate self-complementary regions that form the stem while underlined nucleotides indicate bases that were changed to create base pair mismatches. The 3 templates had different stem and overhang lengths but identical 6-base loop sequences.

Caption: Fig. 2. Extension rate versus mismatch position at 65[degrees]C with template 1 (A) and template 2 (B).

Average rate of 4-6 stopped-flow shots reported. These data are tabulated in Table 2 in the online Data Supplement. Rates decreased for all mismatch types as the mismatch nears the 3' end of the template. Identities of mismatches are shown in the insets. (A), Template 1 mismatches. (B), Template 2 mismatches. Mismatches are identical to those in (A) with the exception that the T * C (gray circles) mismatch was studied instead of the C * T mismatch.

Caption: Fig. 4. Rate vs temperature for several matched and 3' mismatched hairpins.

Error bars represent standard deviation. Identities are shown in the insets. (A), Three matched templates show maximum rates between 68[degrees]C and 70[degrees]C. (B), Six mismatched templates with maximum rates between 55[degrees]C and 60[degrees]C. (C), Mismatch extension rates relative to matched rates increase as temperatures decrease.
Table 1. Apparent Michaelis-Menten parameters. (a,b)

Mismatched bases         []            [K.sub.m]
(primer template)        ([s.sup.-1])           (nmol/L)

Match (template 1)     335 [+ or -] 13       162 [+ or -] 22
G * T (template 1)    4.00 [+ or -] 0.15     256 [+ or -] 28
Match (template 2)      226 [+ or -] 4      60.3 [+ or -] 4.6
G * T (template 2)    3.14 [+ or -] 0.12     176 [+ or -] 26
Match (template 3)      276 [+ or -] 5        110[+ or -]4
G * T (template 3)    8.37 [+ or -] 0.16     348 [+ or -] 17
T * T (template 3)   0.558 [+ or -] 0.018    141 [+ or -] 14

Mismatched bases          []/[K.sub.m]
(primer template)    ([micro][M.sup.-1] [s.sup.-1])

Match (template 1)          2070 [+ or -] 290
G * T (template 1)          15.6 [+ or -] 1.8
Match (template 2)          3740 [+ or -] 300
G * T (template 2)          17.8 [+ or -] 2.7
Match (template 3)          2500 [+ or -] 90
G * T (template 3)          24.1 [+ or -] 1.3
T * T (template 3)         3.96 [+ or -] 0.41

(a) Michaelis/Menten parameters obtained via nonlinear regression of
the observed reaction rate (nt/s) vs concentration data between 10/
1200 nmol/L template as shown in Fig. 5 in the online Data Supplement.

(b) Michaelis-Menten parameters are reported as fit value [+ or -]
standard error.

Table 2. Decision matrix for allele-specific PCR vs melting
analysis for SNV typing.

SNV              SNV
class (a)   frequency (a)       SNV

1               0.662       C/T or G/A

2               0.176       C/A or G/T

3               0.088       C/G

4               0.074       T/A

                            Relative rate at
SNV                           65[degrees]C     [DELTA][T.sub.m]
class (a)   3' mismatches   (% of match) (b)   ([degrees]C) (c)

1               A * C             0.72             0.8-1.4
                T * G             1.60
2               A * G            0.006             0.8-1.4
                T * C             1.01
3               C * C            0.022               <0.4
                G * G            0.114
4               A * A            0.021               <0.4
                T * T            0.140

SNV             Preferred
class (a)         method

1           HRMA (d)

2           AS-PCR or HRMA (d)

3           AS-PCR

4           AS-PCR

a SNV classification and frequency inthe human genome (28).

(b) Ratesare average initial rates of 3' mismatches relative to
a match for templates 1-3 at 65[degrees]C. For relative rate
calculations, "symmetric pairs" of mismatches are equivalent (e.g., A
* C and C * A mismatches are equivalent).

(c) Insilico [DELTA][T.sub.m] calculations (28).

(d) HRMA, high-resolution melting analysis.

Fig. 3. All 3' mismatches with 3 sets of templates at 65[degrees]C.

Extension rates varied 500-fold across mismatched nucleotide pairs.
Asymmetric error bars represent standard deviation of replicate shots.
The increasing size of the error bars is due to a combination of the
logarithmic scale of the graph and increasing CVs for measurements of
slower mismatches (A * A, C * C, G * A, A * G). Template 1 is dark
gray, template 2 is light gray, and template 3 is white.

Extension rate (nt/s/poly)

Match                                   205     190     177

purine*prymidine / pyrimidine*purine
G*T                                     2.50    2.41    4.30
T*T                                     1.17    4.55    2.86
C*A                                     0.686   2.13    2.26
A*C                                     0.500   0.473   2.24

C*T                                     1.75    3.88    3.05
T*C                                     0.326   0.410   2.16
T*T                                     0.091   0.170   0.540
C*C                                     0.061   0.020   0.044

G*G                                     0.353   0.055   0.246
A*A                                     0.036   0.040   0.047
G*A                                     0.009   0.008   0.010
A*G                                     0.013   0.009   0.018

Note: Table made from bar graph.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Rejali, Nick A.; Moric, Endi; Wittwer, Carl T.
Publication:Clinical Chemistry
Date:May 1, 2018
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