- Email: [email protected]
Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing
D J M ram 14 rog 20 E P CM
The Journal of Molecular Diagnostics, Vol. 16, No. 5, September 2014
jmd.amjpathol.org
TECHNICAL ADVANCE Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing Kelli Sumner,* Jeffrey J. Swensen,*y Melinda Procter,* Mohamed Jama,* Whitney Wooderchak-Donahue,*y Tracey Lewis,* Michael Fong,* Lindsey Hubley,* Monica Schwarz,* Youna Ha,* Eleri Paul,* Benjamin Brulotte,z Elaine Lyon,*y Pinar Bayrak-Toydemir,*y Rong Mao,*y Genevieve Pont-Kingdon,*y and D. Hunter Best*y From the ARUP Institute for Clinical and Experimental Pathology,* Salt Lake City; the Department of Pathology,y University of Utah, Salt Lake City; and ARUP Laboratories,z Salt Lake City, Utah CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
Accepted for publication April 23, 2014. Address correspondence to D. Hunter Best, Ph.D., F.A.C.M.G., Department of Pathology, University of Utah School of Medicine, ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108. E-mail: hunter. [email protected].
We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, noneM13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow. (J Mol Diagn 2014, 16: 477e480; http://dx.doi.org/ 10.1016/j.jmoldx.2014.04.005)
Genetic polymorphisms are a potential source of genotyping errors. Most commonly, sequence variants that occur in PCR primer binding sites cause allele-specific PCR dropout, leading to false-negative or apparent homozygous results. However, sequence variants in PCR amplicons can also interfere with mutation detection through other mechanisms. A polymorphism in intron 2 of MEN1, c.-23-16C>G (rs509606), with an allele frequency of approximately 18% in white Europeans, is a common source of genotyping errors in exon 2 of this gene.1 Although rs509606 lies outside the PCR primer binding sites, the allele containing this variant preferentially amplifies in heterozygous individuals, resulting in dropout of the wild-type allele. This amplification Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.04.005
bias has been reported to occur because rs509606 changes the stability of G-quadruplexe and i-motifelike DNA secondary structures in the amplicon, providing a competitive advantage for the variant allele during PCR. Although changing the amplification conditions, as suggested by Wenzel et al,1 represents one solution to this problem, we sought to Supported by institutional funding from the ARUP Institute for Clinical and Experimental Pathology. Disclosures: None declared. Current address of M.F., School of Oceanography, University of Rhode Island, Narragansett, RI; of L.H., College of Pharmacy, University of Utah, Salt Lake City, UT; of M.S., School of Medicine, University of Utah, Salt Lake City, UT.
Sumner et al develop a method that would allow us to maintain consistent PCR conditions between different amplicons to better fit into the workflow of the laboratory. Noncontinuously binding (loop-out) oligonucleotide hybridization probes have been described for molecular haplotyping and multiplex genotyping of nonadjacent sequence variants.2e4 In a probe of this type, stretches of nucleotides are omitted between two or more nearby regions to be tested. Reportedly, flanking segments of 11 nucleotides on either side of the omitted sequence are sufficient for loop-out probes to reliably bind to multiple regions of interest.4 In this study, we used this concept to design noncontinuously binding primers in which nucleotides are omitted (looped-out) during PCR amplification or during Sanger sequencing amplification. We investigated whether loop-out PCR and sequencing primers could be used to exclude regions of problematic DNA sequence, eg, CpG islands or mononucleotide repeats, from PCR amplicons and sequencing amplification reactions for seven genes: argininosuccinate synthase 1 (ASS1), breast cancer 1 (BRCA1, early onset), breast cancer 2 (BRCA2, early onset), methyl CpG binding protein (MECP2), multiple endocrine neoplasia I (MEN1), transforming growth factor beta 1 (TGFBR1), and von Willebrand factor (VWF). Typically, when encountering these problematic regions, several options are available: an additional PCR amplification can be used with different PCR primer sets to get two clean sequencing reads, sequencing can be performed with nested primers, and additives or differing assay conditions can improve results in some cases. Additional PCR and sequencing reactions increase the overall costs of running a test, and using a modified PCR or additives for a single reaction is disruptive to the clinical laboratory workflow.
Materials and Methods Design of Loop-Out Primers Loop-out primers were designed for amplicons in ASS1, BRCA1, BRCA2, MECP2, MEN1, TGFBR1, and VWF Table 1
(Table 1). Primers were designed for three scenarios: i) PCR amplification primers with universal M13 primer tails (MEN1 and ASS1), ii) PCR amplification primers without M13 tails (TGFBR1 and MECP2), and iii) sequencing primers that are placed internally to the PCR primers (BRCA1, BRCA2, and VWF). The looped-out regions were chosen by inspecting M13 sequencing results with traditional primers and identifying problematic regions in the sequencing traces, ie, lowcomplexity repetitive sequence. Primers were then designed to flank these regions. Generally, the higher the GC content of the primer target sequence, the fewer flanking bases are required. The details of oligo design using loop-out technology are described by Pont-Kingdon et al.4 Primer melting temperatures were calculated using the DINAMelt Web Server (http://mfold.rna.albany.edu/?qZDINAMelt/Two-statemelting, last accessed April 8, 2014), which calculates loop-out melting temperatures using a two-state melting (hybridization) program.5 Looped-out bases ranged from 10 to 46 nucleotides, with 8 to 27 nucleotides flanking the looped-out region. Primers ranged from 27 to 40 nucleotides, with melting temperatures that ranged from 53.0 C to 74.2 C. The MEN1 primer was designed to destabilize the G-quadruplexe and i-motifelike sequence by excluding 10 bases, including the rs509606 polymorphism. The ASS1 primers were designed to exclude intronic semirepetitive GC-rich regions of 25 and 28 nucleotides located 50 of exons 9 and 11. The BRCA1, BRCA2, MECP2, TGFBR1, and VWF primers were designed to exclude repetitive regions of 10 to 35 nucleotides.
Comparison between Traditional and Loop-Out Primers Loop-out primers were compared with traditional primers that would amplify through the problematic areas. All the reactions used a standardized amplification and sequencing protocol. The final concentration of each PCR primer was 0.25 mmol/L and of sequencing primers was 0.083 mmol/L. Primers were amplified using FailSafe premixes (Epicentre, Illumina, Madison, WI) and Platinum Taq DNA polymerase
Loop-Out Primers for Amplicons in Selected Genes
Gene
Loop-out primer sequence
Excluded
No. of bases
M13
MEN1 BRCA1 BRCA2
50 -GAACCTGCCCGACC(-)CGGCTTGCCTTGC-30 50 -AGCAAGACTCCATCTC(-)GAAAACAAATGGT-30 50 -CTGGTTAAAACTAAGGTGGG(-)ATTTAGGACCAATAAGTC-30 50 -GCAAGACTCCACCTC(-)CCTGTAGTTCAAC-30 50 -CTATATGTACTATTTAC(-)GACAGAGGTACCTGAATCAGC-30 50 -GCCCATTTGTTCATGTAATC(-)CTGGTATTTTATCTATATTC-30 50 -GTTTAGTGAATTAATAATCC(-)CACAACAAAACCATATTTAC-30 50 -CTCATGGGCACAAT(-)ACATGCCATGTCC-30 50 -CCATTTAAGGCGTTTC(-)TGCAGACTCCTC-30 50 -AAAATGGTGCATGCATTAA(-)ATATTTTCTTG-30 50 -GATGGCCAAACCAGGACATATACTAAA(-)GGAAGGTT-30 50 -GCGAGACTCCATCTC(-)GAAAGAAAAGAATATTC-30
c.-23-15_-23-16 c.441þ19_441þ64 c.68-17_68-1 c.793þ30_793þ64 c.1909þ12_1909þ22 c.2951_2960 c.8633-14_8636 c.567-76_567-52 c.689-86_689-59 c.1156-25_1156-15 c.26þ15_26þ24 c.-2098_-2086
10 46 17 35 11 11 18 25 28 11 10 13
Yes NA NA NA NA NA NA Yes Yes No No NA
ASS1 TGFBR1 MECP2 VWF
Reference sequence NG_008929.1 NG_005905.2 NG_012772.3
NG_011542.1 NC_000009.11 NC_000023.9 NC_000012.11
(-), missing bases; NA, not applicable (sequencing primer).
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The Journal of Molecular Diagnostics
Clinical Advantage of Loop-Out Primers
Figure 1 Sample containing the MEN1 rs509606 polymorphism (not shown); sequences from c.240_259. A: Amplification with a normal forward primer; the 4-bp deletion (black bar), c.249_252delGTCT, was undetectable. B: Amplification with a loop-out forward primer showed a clearly discernable 4-bp deletion, c.249_252delGTCT.
(Life Technologies Inc., Grand Island, NY). Cycling conditions were as follows: 95 C for 5 minutes; 10 cycles of 94 C for 30 seconds, 62 C for 45 seconds with a decrease in temperature by 0.5 C per cycle, and an initial extension at 72 C for 1 minute; this was followed by an additional 25 cycles of 94 C for 30 seconds, 57 C for 45 seconds, and 72 C for 45 seconds; and a final extension at 72 C for 7 minutes. A touchdown PCR protocol was chosen because it increases primer specificity during the early amplification cycles by using higher annealing temperatures, which discourages mispriming of the flanking primer segments.6 The temperature is gradually decreased in each cycle for the first 10 cycles, and then a standard single-temperature profile is used for the remaining cycles. Thus, the specific amplification products should be favored in the earliest cycles and exponentially amplified in the remaining cycles. Sanger sequencing was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems, Life Technologies Inc., Carlsbad, CA) and an Applied Biosystems 3730 DNA analyzer. Sequences were aligned using Mutation Surveyor software version 4.0.6 (SoftGenetics LLC, State College, PA).
Results When a traditional M13-tagged PCR primer design was used for analysis of MEN1 in an individual that carried rs509606
on one allele and a pathogenic mutation in exon 2 (c.249_252delGTCT) on the other allele, preferential amplification of the allele containing the polymorphism was observed (Figure 1A); the mutant allele could easily be overlooked and considered background noise. An M13-tagged loop-out forward PCR primer for exon 2 successfully excluded the region with the rs509606 single nucleotide polymorphism, eliminated allele dropout, and made the mutation clearly discernible (Figure 1B). When exon 9 from the ASS1 gene was amplified with traditional M13-tagged PCR primers, the peak height of the sequence decreased to zero shortly after an intronic G-rich repetitive region (Figure 2). After the intronic repetitive region was excluded using an M13-tagged loop-out forward PCR primer, the sequence from the product was consistent throughout the entire read (Figure 2). Similar loop-out PCR primers without M13 tails also successfully excluded problematic regions in TGFBR1and MECP2 (not shown). BRCA1, BRCA2, and VWF were amplified with traditional PCR primers tagged with M13 tails. However, a custom loop-out sequencing primer was used in one direction of each sequencing reaction; these primers successfully excluded repetitive regions, and the M13 primer used for the other strand allowed the sequence excluded by the loop-out primer to be analyzed (not shown). Loop-out primers were successfully designed to exclude up to 46 nucleotides, with 8 to 27 nucleotides flanking the looped-out region.
Discussion The use of this noncontinuously binding loop-out primer design principle simplifies genetic testing in regions of the genome that are typically difficult to amplify by PCR and/or analyze by Sanger sequencing. These primers can exclude bases in difficult regions that traditionally would require special treatment (eg, additional PCR additives, such as dimethyl sulfoxide or betaine; an annealing temperature different from the rest of the assay; or an additional PCR to obtain two clean
Figure 2 Amplification of ASS1 exon 9. Top: Amplification through the problematic region with a traditional primer. The sequencing signal decreased to zero shortly after the G-rich region. Bottom: Amplification with a loop-out forward primer removed the problematic region, resulting in consistent sequence signal throughout the read.
The Journal of Molecular Diagnostics
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Sumner et al sequence reads from the same strand). In a clinical laboratory setting, keeping assay conditions consistent within and between assays is desirable. Assays that use the same conditions may be batched, which decreases costs, increases throughput, allows for automation, and decreases errors by laboratory personnel. Herein, we show examples of specific uses for loop-out primers. First, allele dropout resulting from the common MEN1 gene polymorphism rs509606 can be prevented through the use of a primer that removes the variant and destabilizes the G-quadruplexe and i-motifelike sequence. This solution preserves the clinically important intronic sequence near the exon and keeps the PCR conditions of this amplicon consistent with the rest of the assay. Second, the GC-rich region in ASS1 that caused sequence peak height to decrease was removed, resulting in consistent signal throughout the entire read. Similar superior results were achieved using the loop-out primer designs in the other genes for PCR amplification with M13 tails, for PCR amplification without M13 tails, and as sequencing primers. We suggest that this design principle can be similarly useful for a wide range of PCR and Sanger
sequencing applications, including removing a common polymorphism located under a PCR primer.
480
jmd.amjpathol.org
References 1. Wenzel JJ, Rossmann H, Fottner C, Neuwirth S, Neukirch C, Lohse P, Bickmann JK, Minnemann T, Musholt TJ, Schneider-Ratzke B, Weber MM, Lackner KJ: Identification and prevention of genotyping errors caused by G-quadruplex- and i-motif-like sequences. Clin Chem 2009, 55:1361e1371 2. Pont-Kingdon G, Chou LS, Damjanovich K, Sumner K, Herrmann M, Erali M, Lyon E: Multiplex genotyping by melting analysis of loci-spanning probes: beta-globin as an example. Biotechniques 2007, 42:193e197 3. Pont-Kingdon G, Lyon E: Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example. Nucleic Acids Res 2005, 33:e89 4. Pont-Kingdon G, Margraf RL, Sumner K, Millson A, Lyon E, Schutz E: Design and application of noncontinuously binding probes used for haplotyping and genotyping. Clin Chem 2008, 54:990e999 5. Markham NR, Zuker M: DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res 2005, 33:W577eW581 6. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS: “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 1991, 19:4008
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The Journal of Molecular Diagnostics
The Journal of Molecular Diagnostics, Vol. 16, No. 5, September 2014
jmd.amjpathol.org
TECHNICAL ADVANCE Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing Kelli Sumner,* Jeffrey J. Swensen,*y Melinda Procter,* Mohamed Jama,* Whitney Wooderchak-Donahue,*y Tracey Lewis,* Michael Fong,* Lindsey Hubley,* Monica Schwarz,* Youna Ha,* Eleri Paul,* Benjamin Brulotte,z Elaine Lyon,*y Pinar Bayrak-Toydemir,*y Rong Mao,*y Genevieve Pont-Kingdon,*y and D. Hunter Best*y From the ARUP Institute for Clinical and Experimental Pathology,* Salt Lake City; the Department of Pathology,y University of Utah, Salt Lake City; and ARUP Laboratories,z Salt Lake City, Utah CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
Accepted for publication April 23, 2014. Address correspondence to D. Hunter Best, Ph.D., F.A.C.M.G., Department of Pathology, University of Utah School of Medicine, ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108. E-mail: hunter. [email protected].
We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, noneM13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow. (J Mol Diagn 2014, 16: 477e480; http://dx.doi.org/ 10.1016/j.jmoldx.2014.04.005)
Genetic polymorphisms are a potential source of genotyping errors. Most commonly, sequence variants that occur in PCR primer binding sites cause allele-specific PCR dropout, leading to false-negative or apparent homozygous results. However, sequence variants in PCR amplicons can also interfere with mutation detection through other mechanisms. A polymorphism in intron 2 of MEN1, c.-23-16C>G (rs509606), with an allele frequency of approximately 18% in white Europeans, is a common source of genotyping errors in exon 2 of this gene.1 Although rs509606 lies outside the PCR primer binding sites, the allele containing this variant preferentially amplifies in heterozygous individuals, resulting in dropout of the wild-type allele. This amplification Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.04.005
bias has been reported to occur because rs509606 changes the stability of G-quadruplexe and i-motifelike DNA secondary structures in the amplicon, providing a competitive advantage for the variant allele during PCR. Although changing the amplification conditions, as suggested by Wenzel et al,1 represents one solution to this problem, we sought to Supported by institutional funding from the ARUP Institute for Clinical and Experimental Pathology. Disclosures: None declared. Current address of M.F., School of Oceanography, University of Rhode Island, Narragansett, RI; of L.H., College of Pharmacy, University of Utah, Salt Lake City, UT; of M.S., School of Medicine, University of Utah, Salt Lake City, UT.
Sumner et al develop a method that would allow us to maintain consistent PCR conditions between different amplicons to better fit into the workflow of the laboratory. Noncontinuously binding (loop-out) oligonucleotide hybridization probes have been described for molecular haplotyping and multiplex genotyping of nonadjacent sequence variants.2e4 In a probe of this type, stretches of nucleotides are omitted between two or more nearby regions to be tested. Reportedly, flanking segments of 11 nucleotides on either side of the omitted sequence are sufficient for loop-out probes to reliably bind to multiple regions of interest.4 In this study, we used this concept to design noncontinuously binding primers in which nucleotides are omitted (looped-out) during PCR amplification or during Sanger sequencing amplification. We investigated whether loop-out PCR and sequencing primers could be used to exclude regions of problematic DNA sequence, eg, CpG islands or mononucleotide repeats, from PCR amplicons and sequencing amplification reactions for seven genes: argininosuccinate synthase 1 (ASS1), breast cancer 1 (BRCA1, early onset), breast cancer 2 (BRCA2, early onset), methyl CpG binding protein (MECP2), multiple endocrine neoplasia I (MEN1), transforming growth factor beta 1 (TGFBR1), and von Willebrand factor (VWF). Typically, when encountering these problematic regions, several options are available: an additional PCR amplification can be used with different PCR primer sets to get two clean sequencing reads, sequencing can be performed with nested primers, and additives or differing assay conditions can improve results in some cases. Additional PCR and sequencing reactions increase the overall costs of running a test, and using a modified PCR or additives for a single reaction is disruptive to the clinical laboratory workflow.
Materials and Methods Design of Loop-Out Primers Loop-out primers were designed for amplicons in ASS1, BRCA1, BRCA2, MECP2, MEN1, TGFBR1, and VWF Table 1
(Table 1). Primers were designed for three scenarios: i) PCR amplification primers with universal M13 primer tails (MEN1 and ASS1), ii) PCR amplification primers without M13 tails (TGFBR1 and MECP2), and iii) sequencing primers that are placed internally to the PCR primers (BRCA1, BRCA2, and VWF). The looped-out regions were chosen by inspecting M13 sequencing results with traditional primers and identifying problematic regions in the sequencing traces, ie, lowcomplexity repetitive sequence. Primers were then designed to flank these regions. Generally, the higher the GC content of the primer target sequence, the fewer flanking bases are required. The details of oligo design using loop-out technology are described by Pont-Kingdon et al.4 Primer melting temperatures were calculated using the DINAMelt Web Server (http://mfold.rna.albany.edu/?qZDINAMelt/Two-statemelting, last accessed April 8, 2014), which calculates loop-out melting temperatures using a two-state melting (hybridization) program.5 Looped-out bases ranged from 10 to 46 nucleotides, with 8 to 27 nucleotides flanking the looped-out region. Primers ranged from 27 to 40 nucleotides, with melting temperatures that ranged from 53.0 C to 74.2 C. The MEN1 primer was designed to destabilize the G-quadruplexe and i-motifelike sequence by excluding 10 bases, including the rs509606 polymorphism. The ASS1 primers were designed to exclude intronic semirepetitive GC-rich regions of 25 and 28 nucleotides located 50 of exons 9 and 11. The BRCA1, BRCA2, MECP2, TGFBR1, and VWF primers were designed to exclude repetitive regions of 10 to 35 nucleotides.
Comparison between Traditional and Loop-Out Primers Loop-out primers were compared with traditional primers that would amplify through the problematic areas. All the reactions used a standardized amplification and sequencing protocol. The final concentration of each PCR primer was 0.25 mmol/L and of sequencing primers was 0.083 mmol/L. Primers were amplified using FailSafe premixes (Epicentre, Illumina, Madison, WI) and Platinum Taq DNA polymerase
Loop-Out Primers for Amplicons in Selected Genes
Gene
Loop-out primer sequence
Excluded
No. of bases
M13
MEN1 BRCA1 BRCA2
50 -GAACCTGCCCGACC(-)CGGCTTGCCTTGC-30 50 -AGCAAGACTCCATCTC(-)GAAAACAAATGGT-30 50 -CTGGTTAAAACTAAGGTGGG(-)ATTTAGGACCAATAAGTC-30 50 -GCAAGACTCCACCTC(-)CCTGTAGTTCAAC-30 50 -CTATATGTACTATTTAC(-)GACAGAGGTACCTGAATCAGC-30 50 -GCCCATTTGTTCATGTAATC(-)CTGGTATTTTATCTATATTC-30 50 -GTTTAGTGAATTAATAATCC(-)CACAACAAAACCATATTTAC-30 50 -CTCATGGGCACAAT(-)ACATGCCATGTCC-30 50 -CCATTTAAGGCGTTTC(-)TGCAGACTCCTC-30 50 -AAAATGGTGCATGCATTAA(-)ATATTTTCTTG-30 50 -GATGGCCAAACCAGGACATATACTAAA(-)GGAAGGTT-30 50 -GCGAGACTCCATCTC(-)GAAAGAAAAGAATATTC-30
c.-23-15_-23-16 c.441þ19_441þ64 c.68-17_68-1 c.793þ30_793þ64 c.1909þ12_1909þ22 c.2951_2960 c.8633-14_8636 c.567-76_567-52 c.689-86_689-59 c.1156-25_1156-15 c.26þ15_26þ24 c.-2098_-2086
10 46 17 35 11 11 18 25 28 11 10 13
Yes NA NA NA NA NA NA Yes Yes No No NA
ASS1 TGFBR1 MECP2 VWF
Reference sequence NG_008929.1 NG_005905.2 NG_012772.3
NG_011542.1 NC_000009.11 NC_000023.9 NC_000012.11
(-), missing bases; NA, not applicable (sequencing primer).
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The Journal of Molecular Diagnostics
Clinical Advantage of Loop-Out Primers
Figure 1 Sample containing the MEN1 rs509606 polymorphism (not shown); sequences from c.240_259. A: Amplification with a normal forward primer; the 4-bp deletion (black bar), c.249_252delGTCT, was undetectable. B: Amplification with a loop-out forward primer showed a clearly discernable 4-bp deletion, c.249_252delGTCT.
(Life Technologies Inc., Grand Island, NY). Cycling conditions were as follows: 95 C for 5 minutes; 10 cycles of 94 C for 30 seconds, 62 C for 45 seconds with a decrease in temperature by 0.5 C per cycle, and an initial extension at 72 C for 1 minute; this was followed by an additional 25 cycles of 94 C for 30 seconds, 57 C for 45 seconds, and 72 C for 45 seconds; and a final extension at 72 C for 7 minutes. A touchdown PCR protocol was chosen because it increases primer specificity during the early amplification cycles by using higher annealing temperatures, which discourages mispriming of the flanking primer segments.6 The temperature is gradually decreased in each cycle for the first 10 cycles, and then a standard single-temperature profile is used for the remaining cycles. Thus, the specific amplification products should be favored in the earliest cycles and exponentially amplified in the remaining cycles. Sanger sequencing was performed using the BigDye terminator cycle sequencing kit (Applied Biosystems, Life Technologies Inc., Carlsbad, CA) and an Applied Biosystems 3730 DNA analyzer. Sequences were aligned using Mutation Surveyor software version 4.0.6 (SoftGenetics LLC, State College, PA).
Results When a traditional M13-tagged PCR primer design was used for analysis of MEN1 in an individual that carried rs509606
on one allele and a pathogenic mutation in exon 2 (c.249_252delGTCT) on the other allele, preferential amplification of the allele containing the polymorphism was observed (Figure 1A); the mutant allele could easily be overlooked and considered background noise. An M13-tagged loop-out forward PCR primer for exon 2 successfully excluded the region with the rs509606 single nucleotide polymorphism, eliminated allele dropout, and made the mutation clearly discernible (Figure 1B). When exon 9 from the ASS1 gene was amplified with traditional M13-tagged PCR primers, the peak height of the sequence decreased to zero shortly after an intronic G-rich repetitive region (Figure 2). After the intronic repetitive region was excluded using an M13-tagged loop-out forward PCR primer, the sequence from the product was consistent throughout the entire read (Figure 2). Similar loop-out PCR primers without M13 tails also successfully excluded problematic regions in TGFBR1and MECP2 (not shown). BRCA1, BRCA2, and VWF were amplified with traditional PCR primers tagged with M13 tails. However, a custom loop-out sequencing primer was used in one direction of each sequencing reaction; these primers successfully excluded repetitive regions, and the M13 primer used for the other strand allowed the sequence excluded by the loop-out primer to be analyzed (not shown). Loop-out primers were successfully designed to exclude up to 46 nucleotides, with 8 to 27 nucleotides flanking the looped-out region.
Discussion The use of this noncontinuously binding loop-out primer design principle simplifies genetic testing in regions of the genome that are typically difficult to amplify by PCR and/or analyze by Sanger sequencing. These primers can exclude bases in difficult regions that traditionally would require special treatment (eg, additional PCR additives, such as dimethyl sulfoxide or betaine; an annealing temperature different from the rest of the assay; or an additional PCR to obtain two clean
Figure 2 Amplification of ASS1 exon 9. Top: Amplification through the problematic region with a traditional primer. The sequencing signal decreased to zero shortly after the G-rich region. Bottom: Amplification with a loop-out forward primer removed the problematic region, resulting in consistent sequence signal throughout the read.
The Journal of Molecular Diagnostics
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jmd.amjpathol.org
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Sumner et al sequence reads from the same strand). In a clinical laboratory setting, keeping assay conditions consistent within and between assays is desirable. Assays that use the same conditions may be batched, which decreases costs, increases throughput, allows for automation, and decreases errors by laboratory personnel. Herein, we show examples of specific uses for loop-out primers. First, allele dropout resulting from the common MEN1 gene polymorphism rs509606 can be prevented through the use of a primer that removes the variant and destabilizes the G-quadruplexe and i-motifelike sequence. This solution preserves the clinically important intronic sequence near the exon and keeps the PCR conditions of this amplicon consistent with the rest of the assay. Second, the GC-rich region in ASS1 that caused sequence peak height to decrease was removed, resulting in consistent signal throughout the entire read. Similar superior results were achieved using the loop-out primer designs in the other genes for PCR amplification with M13 tails, for PCR amplification without M13 tails, and as sequencing primers. We suggest that this design principle can be similarly useful for a wide range of PCR and Sanger
sequencing applications, including removing a common polymorphism located under a PCR primer.
480
jmd.amjpathol.org
References 1. Wenzel JJ, Rossmann H, Fottner C, Neuwirth S, Neukirch C, Lohse P, Bickmann JK, Minnemann T, Musholt TJ, Schneider-Ratzke B, Weber MM, Lackner KJ: Identification and prevention of genotyping errors caused by G-quadruplex- and i-motif-like sequences. Clin Chem 2009, 55:1361e1371 2. Pont-Kingdon G, Chou LS, Damjanovich K, Sumner K, Herrmann M, Erali M, Lyon E: Multiplex genotyping by melting analysis of loci-spanning probes: beta-globin as an example. Biotechniques 2007, 42:193e197 3. Pont-Kingdon G, Lyon E: Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example. Nucleic Acids Res 2005, 33:e89 4. Pont-Kingdon G, Margraf RL, Sumner K, Millson A, Lyon E, Schutz E: Design and application of noncontinuously binding probes used for haplotyping and genotyping. Clin Chem 2008, 54:990e999 5. Markham NR, Zuker M: DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res 2005, 33:W577eW581 6. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS: “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 1991, 19:4008
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The Journal of Molecular Diagnostics