Multiple Heat Pulses during PCR Extension Enabling Amplification of

Technical Note pubs.acs.org/ac

Multiple Heat Pulses during PCR Extension Enabling Amplification of GC-Rich Sequences and Reducing Amplification Bias Arto K. Orpana,† Tho H. Ho,‡ and Jakob Stenman*,‡,§,⊥ †

HUSLAB, Laboratory of Genetics and Department of Medical Genetics, University of Helsinki, Helsinki, Finland Minerva Foundation Institute for Medical Research, Helsinki, Finland § Institute for Molecular Medicine Finland, Helsinki, Finland ⊥ Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden ‡

S Supporting Information *

ABSTRACT: PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.

P

duplexes between the opposite strands of GC-rich sequences, as a result of either incomplete denaturation or reannealing, when the reaction temperature decreases toward the annealing step.14 In addition, strong secondary structures can form as a result of intramolecular folding of the separate DNA strands.15−18 Such secondary structures are thought to block DNA polymerase activity during the extension step, resulting in incomplete extension products, low extension efficiency, and ultimately, inefficient amplification of the specific products. As a result, amplification of nonspecific products occurs more commonly in PCR amplification of GC-rich sequences despite attempts at optimizing cycling protocols.19 In contrast to almost all existing approaches that focused on optimizing the annealing step, we aimed at altering the extension step in order to improve the amplification efficiency over long GC-rich and/or repetitive sequences. More specifically, we introduced multiple heat pulses to the extension step to generate a novel PCR cycling protocol for amplification of difficult target sequences with a high GC-content. We hypothesized that these heat pulses would temporarily

olymerase chain reaction (PCR) has been used for more than two decades, and new applications and modifications emerge every day. Although PCR protocols can be optimized for amplification of most DNA or cDNA templates, there are still situations when certain target templates cannot be amplified using conventional cycling protocols. Various improvements have been developed for PCR amplification of extremely GC-rich and/or long repetitive sequences. These can be categorized into reagent development and optimization of cycling protocols. Reagents developed for improving amplification of GC-rich sequences include DNA polymerase blends;1,2 various additives, cosolvents, such as DMSO,3 glycerol, and betaine;3,4 modified nucleotides, like 7-methyl dGTP5 or 7-deaza dGTP;5,6 chemical modification of the template DNA;7 or combinations thereof.8 In addition, several PCR cycling protocols such as “Two-Step hot PCR”;9 “Slow down PCR”;10,11 and “Hotstart and Touchdown PCR”12,13 have been designed for this purpose. In spite of these improvements, amplification of long, extremely GC-rich and/ or repetitive fragments still remains challenging and in some cases impossible. Secondary structures with high melting temperatures are considered to be the major obstacle blocking the DNA polymerase from completing the extension of annealed primers during PCR.14−16 Such secondary structures can arise from © 2012 American Chemical Society

Received: November 16, 2011 Accepted: January 5, 2012 Published: January 5, 2012 2081

dx.doi.org/10.1021/ac300040j | Anal. Chem. 2012, 84, 2081−2087

Analytical Chemistry

Technical Note

Figure 1. Illustrative temperature profile for each cycle of the Heat Pulse Extension PCR cycling protocol (HPE-PCR). After initial denaturation, 35 cycles of PCR amplification was performed with a two-stage denaturation step (94 °C for 45 s and 98° for 10 s), 30 s annealing at the templatespecific estimated optimal annealing temperature, followed by gradual heating to a basal extension temperature in the range of 72−80 °C and multiple heat pulses with a peak temperature in the range of 80−90 °C during the extension step.

specific estimated optimal annealing temperature for 30 s. The conventional cycling protocols for amplification of the Fragile X sequences were performed with extension steps at 72, 78, or 83 °C for comparison with the HPE-PCR protocol. Other recently reported PCR cycling protocols for amplification of GC-rich sequences, including Slowdown PCR,11 Hotstart and Touchdown PCR,12 and SAFE PCR13 were also performed as described in the original reports with a modified extension time of 2 min to amplify the sequences of up to 3 kb in length (the extension time of EXT DNA polymerase recommended by manufacturer is 40 s/kb). Agarose Gel Electrophoresis. Ten microliters of PCR products in a mixture with 2 μL of 6× loading dye (Fermentas) was separated on 1% agarose gels in TBE buffer with a GeneRuler 1 kb DNA Ladder (Fermentas) and run at 110 V until sufficient separation was achieved. Gels were stained with the SYBR Gold Nucleic Acid Gel Stain (10 000×, Invitrogen) and imaged by AlphaImagerMini system (Alpha Innotech). DNA Sequencing and Analysis. PCR products were excised from 1% agarose gel and purified by a Nucleospin Extract II gel extraction kit (MACHEREY-NAGEL GmbH & Co.KG). Sequencing of PCR products was commercially provided by the Institute for Molecular Medicine, Finland (FIMM, P.O. Box 20, FI-00014 University of Helsinki, Finland). DNA alignments were conducted with BLAST/ blastn application from NCBI. Different Fragile X expansions amplified by HPE-PCR were also digested with the EcoN1 enzyme (New England Biolab) according to the recommendation from manufacturer. This restriction enzyme realizes and cuts the double-stranded DNA containing the sequence 5′...CCTNNNNNAGG...3′ that is present in all Fragile X expansion amplicons in this study, resulting in a fragment of about 110 bp and the other fragment of variable length depending on the number of expansion repeats.

destabilize secondary structures, in order to improve DNA polymerase extension and subsequently increase efficiency and specificity of amplification of such sequences.

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EXPERIMENTAL SECTION DNA Samples. Human genomic DNA was extracted from 200 μL of healthy whole blood using a QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany). Fragile X cell line DNA samples, kindly provided by Finnzymes Oy (Vantaa, Finland), were obtained from Coriell Cell Repositories (Coriell Institute for Medical Research). All DNA samples were quantified with a NanoVue spectrophotometer (GE Healthcare, Waukesha, WI) and diluted to 10 ng/μL in distilled H2O, prior to the PCR. PCR Components. The PCR master mix for amplification of Fragile X sequences consisted of 0.8× PCR buffer, 0.16 mM dATP, 0.16 mM dTTP, 0.56 mM dCTP, 0.56 mM dGTP, forward and reverse primers 1.5 μM, Betaine 1.8 M (SigmaAldrich, St. Louis, MO), DyNAzyme EXT DNA polymerase 0.12 U/μL (Finnzymes Oy, Vantaa, Finland), and 20 ng of template DNA, in a total reaction volume of 20 μL. Betaine is omitted in PCR master mix without additive, and DMSO (5%) replaces Betaine in the PCR master mix with DMSO (5%). The PCR master mix for amplification of GNAQ and GP1BB sequences consisted of 1× PCR buffer, 0.2 mM dNTP, the forward and reverse primers 0.4 μM, DyNAzyme EXT DNA polymerase 0.03 U/μL (Finnzymes, Vantaa, Finland), and 20 ng of template DNA, in a total reaction volume of 20 μL. Primer Design. Primer design was performed by commercial software (Oligo primer analysis software 6.8, Molecular Biology Insights, Inc., USA). All primers were ordered from TAG Copenhagen A/S. PCR Cycling Conditions. PCR amplification was performed on a 24-well Piko thermal cycler (Thermo Fisher Scientific Oy, Vantaa, Finland), with different cycling protocols, the conventional PCR protocols with a constant extension temperature, and the Heat Pulse Extension PCR protocol (HPE-PCR) as listed in Table 2 and described further in the Results and Discussion section. Both cycling protocols begin with an initial denaturation step at 94 °C for 7 min, followed by 35 cycles with a two-stage denaturation step (94 °C for 45 s and 98 °C for 10 s) and a primer annealing step at a template-

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RESULTS AND DISCUSSION Overview of Cycling Protocol with Heat Pulse Extension. In each cycle of the HPE-PCR protocol, a twostage denaturation step (94 °C for 45 s and 98° for 10 s) was followed by a primer-annealing step at a template-specific estimated optimal temperature. Unlike conventional PCR 2082



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Table 1. Primer Sequences gene

primer

sequence

GNAQ

GNAQF1 GNAQR1 GNAQF2 GNAQR2 GPIBF1 GPIBR1 GPIBF2 GPIBR2 FrXF

5′-CAGCACGCCATGATGGACTC-3′ 5′-GGGAGGGCTCGCACTGA-3′ 5′-AGGCCATCCCCCAACGTG-3′ 5′-AGGAGCGACCCCGTGGAG-3′ 5′-CGTGCGGGGTGGTCAGG-3′ 5′-GAGTTTGCAGGCCCGTGTTG-3′ 5′-TGAGACCCCATTTTCTGTCGAG-3′ 5′-GGGGAATTTCCAGGTCAGTCGT-3′ 5′-TCACCGCCCTTCAGCCTTCCCGCCCTCCAC-3′

FrXR

5′-GGGCCTGCCTCCCGCCGACACCAA-3′

GP1BB

FMR1

product length 706 bp 1893 bp 822 bp 1890 bp 30 repeats 95 repeats 140 repeats 200 repeats 336 repeats 500 repeats 550 repeats 930 repeats

0.5 kb 0.7 kb 0.8 kb 1 kb 1.4 kb 1.9 kb 2.1 kb 3.2 kb

Table 2. Temperature Profile of the HPE-PCR Cycling Protocol 35 cycles extension with heat pulses initial denaturation

denaturation

annealing

94 °C × 7 min

95° × 45 s 98° × 10 s

61° × 30 s

ramp to 78° (0.6 °C/s)

7×

86° × 2 s 78° × 2 s

7×

90° × 2 s 78° × 2 s

subcycling during each extension step, including both the extension time at a basal temperature and the time necessary for destabilizing thermostable structures at a higher peak temperature. In addition, heating and cooling during subcycling causes a delay that is dependent on the maximal ramp rate of the thermal cycler used. Multiple Heat Pulses in the Extension Step Allow Amplification over Long CGG-Repeat Expansions in Fragile X Syndrome. Fragile X syndrome is caused by the expansions of CGG repeats in the 5′-untranslated region of the X chromosomal Fragile mental retardation 1 (FMR1) gene.20 The Fragile X sites containing more than 200 repeats are fullmutation expansions that cause abnormal methylation21 and typical Fragile X syndrome.22 Expansions spanning from 55 to 200 repeats are considered as premutation expansions that would relate to other syndromes23,24 and might lead to development of full-mutation expansions in the next generations.25,26 Although PCR seems to be a high-throughput, fast, and simple method for sizing analysis of these expansions, amplification over these sequences is challenging. The 100%GC content and the self-complementary nature of these sequences lead to the formation of very stable hairpin structures.17,18 In addition, it is even more challenging to amplify the full-mutation expansions in the presence of unexpanded alleles. The shorter wild type alleles tend to be extended with a higher efficiency during PCR, thus finally dominating the amplification reactions and hindering the amplification of the full-mutation expansion.27 Currently, there is no optimal PCR program that is capable of amplifying CGG-repeat expansions longer than 330 repeats in male samples or 160 repeats in carrier female samples, using standard PCR reagents despite introduction of modified nucleotides.27−34 In a recent study, amplification of longer Fragile X expansions was demonstrated using a nondisclosed set of PCR

cycling protocols where the extension temperature remains constant, multiple heat pulses were introduced in the extension step. The extension step starts by gradual heating to a basal extension temperature in the range of 72−80 °C and is followed by multiple heat pulses (Figure 1). In each heat pulse, the reaction temperature is increased to a peak temperature in the range of 80−90 °C and, then, quickly drops back to the basal extension temperature before the next heat pulse. The number of heat pulses needed for efficient amplification is dependent on the length, sequence, and GC-content of the DNA template. In this study, we used 14 pulses, which was sufficient for all tested sequences. While 72 °C is commonly regarded as the optimal extension temperature for many thermostable DNA polymerase enzymes, there is considerable sequence-specific variation in the optimal temperature for destabilizing secondary structures and highmelting-temperature duplexes. As a result, the DNA polymerase is in many cases inhibited or completely prevented by such secondary structures14,15 when extension is performed at a constant temperature, optimal for enzyme activity. In the HPEPCR protocol, multiple heat pulses in the range of 80−90 °C were introduced with increasing stringency toward the end of the extension step. The peak temperatures used in these heat pulses were clearly above the optimal temperature for DNA polymerase activity. Complete dissociation of partly extended strands from their templates was prevented by keeping the pulse period relatively short (Hold 0−2 s) and positioning primers several hundred bases away from the difficult GC-rich regions. Accordingly, the heating and cooling ramp rates of the heat pulses were set at a maximum (7 °C/s). The HPE-PCR cycling protocol typically takes 4−6 h to run, depending on the number of heat pulses introduced in each extension step. This run time is comparable to Slowdown PCR protocol, and it is considerably longer than conventional PCR cycling protocols. The increased cycling time is caused by 2083



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reagents that were selected by the screening of more than 60 distinct primer pairs and about 1000 combinations of different PCR additives and other buffer components, as well as optimization of PCR cycling protocol.35 We have used a panel of Fragile X cell lines containing different expansions with a known number of repeats, spanning from 23 to 940 CGG repeats, as a model to investigate the performance of the HPEPCR cycling protocol using standard PCR reagents. (See the Experimental Section.) Primers used in these experiments are listed in Table 1. As shown in Figures 2A and S1−5 (Supporting Information), the HPE-PCR cycling protocol enabled specific PCR amplification of all full-mutation expansions in the tested Fragile X cell lines. The longest expansion contains 940 CGG repeats (NA09237), corresponding to a 2.8 kb segment of 100% GC. In comparison, a conventional PCR protocol with a constant extension temperature at 72 °C for 2 min failed to amplify the expansion of 500 repeats and that of 940 repeats in homozygous cell lines (Figure 2B: lanes 1, 2). At the same time, multiple unexpected bands of nonspecific products appeared, which are consistent with previous reports regarding the tendency to form nonspecific products during PCR amplification of GC-rich sequences.19 The corresponding PCR protocol with an elevated extension temperature at 78 °C shows yielded improvement; however, the largest expansion of 940 repeats could not be amplified, and nonspecific products were still formed (Figure-S6A, in Supporting Information). When the extension temperature increased further to 83 °C, no PCR product of the expanded alleles was formed and only a faint band for each wild type unexpanded allele was detected (Figure-S6B, in Supporting Information), indicating low extension efficiency at such high temperature. Since the denaturation and annealing steps of the HPE-PCR cycling protocol is identical to those of the corresponding PCR programs with constant extension temperature, we conclude that the improved performance of the HPE-PCR protocol is a result of improved extension over difficult GC-rich segments. Moreover, we were able to successfully amplify both the large expansions and the wild type-unexpanded allele in different heterozygous cell lines (NA07537, NA06896, NA20239) using the HPE-PCR cycling protocol (Figure 2A: lanes 3, 5, 6). In contrast, all amplification reactions in which a conventional cycling protocol was used showed marked amplification bias favoring the considerably shorter wild type allele. This resulted in a complete lack of amplification of the expanded alleles in all of the samples from heterozygous cell lines (Figure 2B: lanes 3, 5, 6 and Figure-S6A,B: lanes 3, 5, 6, in Supporting Information). This improved performance of the HPE-PCR cycling protocol might be explained by an increased rate of complete extension of the expanded alleles during each cycle of PCR, thus improving the balance of amplification efficiencies between wild type and expanded sequences. In order to verify the performance of the HPE-PCR technique, we conducted a comparison to other recently reported PCR cycling protocols for amplification of GC-rich sequences using the exactly same reagents. While we were able to amplify Fragile X expansions containing up to 940 CGG repeats using the HPE-PCR method, we could only amplify the short wild type allele of about 30 CGG repeats using the “SAFE PCR” protocol and failed to amplify the expansions of 95 CGG repeats or more (Figure 2D: lanes 1, 2, 3, 5, 6). Both the “Slow down PCR” and “Hotstart and Touchdown PCR” protocol even failed to amplify the short wild type-unexpanded alleles

Figure 2. Amplification of Fragile X site from cell lines with different PCR programs. (A) HPE-PCR cycling protocol; (B) conventional PCR; (C) slowdown PCR; (D) SAFE PCR. Coriell Cell Repositories (CCR) catalog numbers are shown in the bottom, followed by the CRC-provided CGG repeat number (in parentheses). The repeat numbers of expanded alleles that could only be amplified by HPE-PCR are shown in red. The expected sizes of PCR products calculated from the CGG-repeat numbers for each cell line are listed as follows: lane 1 (NA09237): 3.2 kb; lane 2 (NA07862): 1.9 kb and 2.1 kb; lane 3 (NA07537): 1.4 kb and 0.5 kb; lane 4 (NA20238): 0.5 kb; lane 5 (NA06896): 0.8 kb, 0.7 kb, and 0.5 kb; lane 6 (NA20239): 1 kb and 0.5 kb. 2084



Technical Note

PCR master mix was set up according to the recommendation from the manufacturer (see the Experimental Section). The same PCR program with heat pulse extension that had been used for the previous experiments was applied to amplify all these segments. As shown in Figures 3B and S7−10 (Supporting Information), a specific amplification product with the expected size and precise sequence could be detected for each primer pair, even when no additives or modified nucleotides were included in the PCR reaction. For comparison, in a previous study describing the “Slowdown PCR” technique for amplification of extremely GC-rich sequences, the GC-rich regions of the GNAQ gene could only be amplified after subdividing the sequence into smaller targets of less than 350 bps. In addition, this method often requires additives and modified nucleotides to amplify sequences with GC-contents higher than 75%.11 It is also worthy to note that the HPE-PCR protocol was capable of amplifying long GC-rich amplicons with several local regions that are not GC-rich, while PCR methods optimized for amplification of GC-rich sequences might not work for sequences with a lower GC content. To the best of our knowledge, almost all current PCR program optimization approaches for amplification of GC-rich sequences have focused on optimization of the annealing step.9−12 The increased performance gained by this approach might stem from the preference of specific over nonspecific priming, which in turn will compensate, to some extent, for low extension efficiency over difficult target sequences. However, in situations where the target sequences have a tendency of forming exceptionally stable secondary structures, this approach is often insufficient. In addition, it is reasonable to assume that such preference of specific over nonspecific priming would not

(Figure 2C and Figure-S6C, Supporting Information), indicating the incompatibility of these protocols with standard PCR reagents used in the HPE-PCR method. This data is consistent with the lack of previous reports of successful amplification of Fragile X expansions longer than 330 CGG repeats in male samples and longer than 160 CGG repeats in female samples using standard PCR reagents. Although the HPE-PCR cycling protocol showed superior performance over conventional PCR and other optimized PCR methods, we were not able to amplify the longest expanded Fragile X alleles (940 repeats) without addition of Betaine cosolvent (Figure 2A: lane WO). There was, however, no corresponding favorable effect from addition of the more commonly used DMSO (5%) (Figure 2A: lane DMSO). The possible mechanism might result from the betaine’s isostabilizing effect36−38 on both the DNA and the DNA polymerase molecules during the extension step. At this stage, we have not extensively studied the possible beneficial effect of any other additives and their combinations. However, in light of the recent study by Filipovic-Sadic et al., it seems reasonable to continue exploring this path in order to further increase the capacity of the HPE-PCR cycling protocol for amplifying very long GC-rich sequences.35 Amplification of Long Nonrepetitive GC-Rich Sequences. In order to assess the versatility of the HPE-PCR cycling protocol for amplification of long GC-rich sequences, we also examined the capability of this technique by amplifying nonrepetitive GC-rich segments on the GNAQ and GP1BB genes. Two primer pairs were designed for each gene (Table 1) to amplify the ∼700 bp and ∼2 kb fragments that cover the highly GC-rich regions depicted in Figure 3A. The standard

Figure 3. Amplification of nonrepetitive GC-rich sequences using HPE-PCR cycling protocol. (A) GC content of GNAQ and GP1BB sequences. The local percentages of GC for each sequence were calculated in every 50 nucleotide window by FastPCR software (PrimerDigital Ltd., Finland). The ∼700 bp regions and ∼2 kb regions that would be amplified by the novel PCR program on each gene are marked with the dashed lines and the solid lines, respectively. (B) Agarose gel electrophoresis. Ten microliters of PCR products were separated on 1% agarose gels. The expected sizes of different amplicons are listed as follows: GP1BB-long: 1.9 kb; GP1BB-short: 822 bp; GNAQ-short: 706 bp; GNAQ-long: 1.9 kb. 2085



Technical Note

(4) Henke, W.; Herdel, K.; Jung, K.; Schnorr, D.; Loening, S. A. Nucleic Acids Res. 1997, 25, 3957−3958. (5) Dierick, H.; Stul, M.; De Kelver, W.; Marynen, P.; Cassiman, J. J. Nucleic Acids Res. 1993, 21, 4427−4428. (6) Fernandez-Rachubinski, F.; Eng, B.; Murray, W. W.; Blajchman, M. A.; Rachubinski, R. A. DNA Sequence 1990, 1, 137−140. (7) Horiuchi, H.; Osawa, M.; Furutani, R.; Morita, M.; Tian, W.; Awatsu, Y.; Shimazaki, H.; Umetsu, K. Genet. Test. 2005, 9, 328−333. (8) Musso, M.; Bocciardi, R.; Parodi, S.; Ravazzolo, R.; Ceccherini, I. J. Mol. Diagn. 2006, 8, 544−550. (9) Schuchard, M.; Sarkar, G.; Ruesink, T.; Spelsberg, T. C. Biotechniques 1993, 14, 390−394. (10) Bachmann, H. S.; Siffert, W.; Frey, U. H. Pharmacogenetics 2003, 13, 759−766. (11) Frey, U. H.; Bachmann, H. S.; Peters, J.; Siffert, W. Nat. Protoc. 2008, 3, 1312−1317. (12) Hube, F.; Reverdiau, P.; Iochmann, S.; Gruel, Y. Mol. Biotechnol. 2005, 31, 81−84. (13) Wei, M.; Deng, J.; Feng, K.; Yu, B.; Chen, Y. Anal. Chem. 2010, 82, 6303−6307. (14) McDowell, D. G.; Burns, N. A.; Parkes, H. C. Nucleic Acids Res. 1998, 26, 3340−3347. (15) Chou, Q. Nucleic Acids Res. 1992, 20, 4371. (16) Usdin, K.; Woodford, K. J. Nucleic Acids Res. 1995, 23, 4202− 4209. (17) Chen, X.; Mariappan, S. V.; Catasti, P.; Ratliff, R.; Moyzis, R. K.; Laayoun, A.; Smith, S. S.; Bradbury, E. M.; Gupta, G. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 5199−5203. (18) Darlow, J. M.; Leach, D. R. J. Mol. Biol. 1998, 275, 3−16. (19) Mamedov, T. G.; Pienaar, E.; Whitney, S. E.; TerMaat, J. R.; Carvill, G.; Goliath, R.; Subramanian, A.; Viljoen, H. J. Comput. Biol. Chem. 2008, 32, 452−457. (20) Verkerk, A. J.; Pieretti, M.; Sutcliffe, J. S.; Fu, Y. H.; Kuhl, D. P.; Pizzuti, A.; Reiner, O.; Richards, S.; Victoria, M. F.; Zhang, F. P.; et al. Cell 1991, 65, 905−914. (21) Oberle, I.; Rousseau, F.; Heitz, D.; Kretz, C.; Devys, D.; Hanauer, A.; Boue, J.; Bertheas, M. F.; Mandel, J. L. Science 1991, 252, 1097−1102. (22) Maddalena, A.; Richards, C. S.; McGinniss, M. J.; Brothman, A.; Desnick, R. J.; Grier, R. E.; Hirsch, B.; Jacky, P.; McDowell, G. A.; Popovich, B.; Watson, M.; Wolff, D. J. Genet. Med. 2001, 3, 200−205. (23) Hagerman, R. J.; Leehey, M.; Heinrichs, W.; Tassone, F.; Wilson, R.; Hills, J.; Grigsby, J.; Gage, B.; Hagerman, P. J. Neurology 2001, 57, 127−130. (24) Allingham-Hawkins, D. J.; Babul-Hirji, R.; Chitayat, D.; Holden, J. J.; Yang, K. T.; Lee, C.; Hudson, R.; Gorwill, H.; Nolin, S. L.; Glicksman, A.; Jenkins, E. C.; Brown, W. T.; Howard-Peebles, P. N.; Becchi, C.; Cummings, E.; Fallon, L.; Seitz, S.; Black, S. H.; ViannaMorgante, A. M.; Costa, S. S.; Otto, P. A.; Mingroni-Netto, R. C.; Murray, A.; Webb, J.; Vieri, F.; et al. Am. J. Med. Genet. 1999, 83, 322− 325. (25) Nolin, S. L.; Brown, W. T.; Glicksman, A.; Houck, G. E. Jr.; Gargano, A. D.; Sullivan, A.; Biancalana, V.; Brondum-Nielsen, K.; Hjalgrim, H.; Holinski-Feder, E.; Kooy, F.; Longshore, J.; Macpherson, J.; Mandel, J. L.; Matthijs, G.; Rousseau, F.; Steinbach, P.; Vaisanen, M. L.; von Koskull, H.; Sherman, S. L. Am. J. Hum. Genet. 2003, 72, 454− 464. (26) Fernandez-Carvajal, I.; Lopez Posadas, B.; Pan, R.; Raske, C.; Hagerman, P. J.; Tassone, F. J. Mol. Diagn. 2009, 11, 306−310. (27) Haddad, L. A.; MingroniNetto, R. C.; ViannaMorgante, A. M.; Pena, S. D. J. Hum. Genet. 1996, 97, 808−812. (28) Baskaran, N.; Kandpal, R. P.; Bhargava, A. K.; Glynn, M. W.; Bale, A.; Weissman, S. M. Genome Res. 1996, 6, 633−638. (29) Brown, W. T.; Nolin, S.; Houck, G. Jr.; Ding, X.; Glicksman, A.; Li, S. Y.; Stark-Houck, S.; Brophy, P.; Duncan, C.; Dobkin, C.; Jenkins, E. Am. J. Med. Genet. 1996, 64, 191−195. (30) Hamdan, H.; Tynan, J. A.; Fenwick, R. A.; Leon, J. A. Mol. Diagn. 1997, 2, 259−269.

improve the balance of amplification efficiencies between PCR templates of different length and GC composition in a heterogeneous sample. Since all templates are primed with the same efficiency, the shorter/less GC-rich sequences will be extended more efficiently, regardless of optimization of priming specificity in the annealing step. In contrast, the HPE-PCR cycling protocol aims specifically at improving the efficiency of extension step and, thus, minimizes the differences in amplification efficiencies between target templates of different length and GC-content. As a result, it not only allows amplification over extremely GC-rich and/or repetitive sequences but also improves the balance of amplification efficiencies between PCR-templates with different length and GC-content. Although not examined in depth in this study, the latter capability might be useful in quantitative competitive PCR, multiplex PCR, bisulfite-based cytosine methylation analysis, and different applications related to amplification of nucleic acid libraries, such as library preparation for next generation sequencing, aptamer technology, and environmental microbiology studies that have been reported to be hampered by amplification bias.39−43

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CONCLUSION In this paper, we present a novel PCR cycling protocol for amplification of long, extremely GC-rich, and/or repetitive sequences. This technique utilizes multiple heat pulses during the extension step in every PCR cycle to destabilize secondary structures caused by intramolecular folding of the DNA template and reannealing of PCR products. This improves the extension efficiency of long GC-rich sequences and reduces the amplification bias that favors amplification of shorter and/ or less GC-rich products. As a result, this technique improves the amplification balance between template sequences of different length and enables amplification of allelic expansions in the presence of a significantly shorter wild type allele.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected] Fax: +46-8-51777712.

ACKNOWLEDGMENTS This work was supported by Finska Läkare Sällskapet and Minerva Foundation for Medical Research. The technical development was done in collaboration with Expression Analytics Oy. Fragile X cell line samples and reagents were kindly provided by Finnzymes Oy, part of Thermo Fisher Scientific. We thank Eeva Lehto for excellent technical assistance and Prof. Vesa Olkonen, Dr. Olivier Béaslas, and Dr. Tero Viitanen for their helpful discussion and providing us the restriction enzyme EcoNI. A.K.O. and T.H.H. contributed equally to this work.

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REFERENCES

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