Locking of 3′ ends of single-stranded DNA templates for improved Pyrosequencing™ performance
Self-annealing of single-stranded template was also the problem with the determination of allele frequency of SNP rs226379 in the A2M gene ((Figure 3)). A ghost peak at the SNP determining position ((Figure 3)B) falsified the calculation of allele frequency in a pooled sample, resulting in a minor allele frequency of about 8% ((Figure 3)C). However, after locking 3′ ends of the single-stranded template by the A2M blOligo or by TdT treatment, the PCR fragments indicate no self-priming, and the calculation of the minor allele frequency results in about 22%, which is in much closer agreement with the National Center for Biotechnology Information Single Nucleotide Polymorphism Database (NCBI) dbSNP; http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=226379) (average allele frequency within a Caucasian sample set of 125 individuals of the C allele: 0.34) ((Figure 3), D and E).
Determination of allele frequencies of the A2M gene single nucleotide polymorphism (SNP) rs226379 in pooled genomic DNA. The sequence around SNP rs226379 was analyzed by the Pyrosequencing of a single sample containing pooled genomic DNA. (A) Theoretical histograms for the genotypes determined by SNP rs226379. The positions of the bases informative for the SNP are highlighted. (B) Pyrogram of a blank control experiment of single-stranded template without sequencing primer. (C) Pyrogram obtained using the standard protocol. (D) Pyrogram obtained using the α-2-macroglobulin (A2M) blOligo (blocking oligonucleotide). (E) Pyrogram using ddCMP-modified template. Red arrows indicate allele-specific base. The blue insets show the allele frequencies calculated from the peak heights.
In summary, we demonstrated that locking the 3′ end of single-stranded templates considerably improves the accuracy of allele calling by Pyrosequencing. It also allows the user to design PCR primers more or less independently of the methodology of Pyrosequencing. This is highly advantageous when a PCR has been already established in mutational profiling of a genomic region because the respective primer pairs from the screening can be used immediately for Pyrosequencing (with one of the primers being biotinylated). Thus, a special PCR design for Pyrosequencing is not necessary. Both the blOligo and TdT treatment gave similar results, and the method chosen may depend on different parameters.
The blOligo has to be custom-made for each kind of template. However, it has the advantage of no additional hands-on time during the sequencing process because it anneals together with the sequencing primer. The principle of locking the 3′ end by a blOligo and avoiding self-priming of the template can also be applied to the destruction of secondary structures of single-stranded templates (e.g., hairpins). Moreover, an additional blOligo could be annealed a few bases downstream of the SNP under investigation to prevent the formation of any secondary structures.
On the other hand, TdT treatment is universally applicable. However, it needs additional hands-on time for incubation. Nevertheless, as a major advantage, it will block the 3′ end of any DNA or oligonucleotide in the reaction mixture. Thus, the TdT treatment will avoid even the falsifying background signals generated by incomplete or recombinant PCR products (13) in pooled genomic or cDNA samples, occurring even if the blOligo is added because these molecules lack the 3′ end targeted by the blOligo. In such cases, and in particular for bisulfite-treated DNA, which is hampered by its lower complexity, the TdT treatment will be the method of choice.
In conclusion, the two methods presented for blocking of ssDNA 3′ ends are simple and reliable approaches for avoiding misinterpretation in genotyping, quantifying allelic imbalance or allele frequencies, and sequencing when using Pyrosequencing.
1.) Ehn M. Ahmadian A. Nilsson P. Lundeberg J. Hober S., Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing, Electrophoresis, P3289 - P3299
2.) Nordstrom T. Alderborn A. Nyren P., Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing technology, J. Biochem. Biophys. Methods, P71 - P82
3.) Ronaghi M. Pettersson B. Uhlen M. Nyren P., PCR-introduced loop structure as primer in DNA sequencing, BioTechniques, P876 - P884
4.) Nordstrom T. Nourizad K. Ronaghi M. Nyren P., Method enabling pyrosequencing on double-stranded DNA, Anal. Biochem., P186 - P193
5.) Andreasson H. Asp A. Alderborn A. Gyllensten U. Allen M., Mitochondrial sequence analysis for forensic identification using pyrosequencing technology, BioTechniques, P124 - P133
6.) Garcia A. C. Ahmadian A. Gharizadeh B. Lundeberg J. Ronaghi M. Nyren P., Mutation detection by pyrosequencing: sequencing of exons 5–8 of the p53 tumor suppressor gene, Gene, P249 - P257
7.) Nordstrom T. Gharizadeh B. Pourmand N. Nyren P. Ronaghi M., Method enabling fast partial sequencing of cDNA clones, Anal. Biochem., P266 - P271
8.) Gharizadeh B. Ghaderi M. Donnelly D. Amini B. Wallin L. K. Nyren P., Multiple-primer DNA sequencing method, Electrophoresis, P1145 - P1151
9.) Rickert M. A. Premstaller A. Gebhardt C. Oefner J. P., Genotyping of SNPs in a polyploid genome by pyrosequencing, BioTechniques, P592 - P600
10.) Ronaghi M., Improved performance of pyrosequencing using single-stranded DNA-binding protein, Anal. Biochem., P282 - P288
11.) Bhattacharya S. Botuyan V. M. Hsu F. Shan X. Arunkumar I. A. Arrowsmith H. C. Edwards M. A. Chazin J. W., Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A, Protein Sci., P2316 - P2325
12.) Yan H. Yuan W. Velculescu E. V. Vogelstein B. Kinzler W. K., Allelic variation in human gene expression, Science, P1143
13.) Judo S. M. Wedel B. A. Wilson C., Stimulation and suppression of PCR-mediated recombination, Nucleic Acids Res., P1819 - P1825