An improved allele-specific PCR primer design method for SNP marker analysis and its application
Although Single Nucleotide Polymorphism (SNP) marker is an invaluable tool for positional cloning, association study and evolutionary analysis, low SNP detection efficiency by Allele-Specific PCR (AS-PCR) still restricts its application as molecular marker like other markers such as Simple Sequence Repeat (SSR). To overcome this problem, primers with a single nucleotide artificial mismatch introduced within the three bases closest to the 3’end (SNP site) have been used in AS-PCR. However, for one SNP site, nine possible mismatches can be generated among the three bases and how to select the right one to increase primer specificity is still a challenge.
In this study, different from the previous reports which used a limited quantity of primers randomly (several or dozen pairs), we systematically investigated the effects of mismatch base pairs, mismatch sites and SNP types on primer specificity with 2071 primer pairs, which were designed based on SNPs from Brassica oleracea 01-88 and 02-12. According to the statistical results, we (1) found that the primers designed with SNP (A/T), in which the mismatch (CA) in the 3rd nucleotide from the 3’ end, had the highest allele-specificity (81.9%). This information could be used when designing primers from a large quantity of SNP sites; (2) performed the primer design principle which forms the one and only best primer for every SNP type. This is never reported in previous studies. Additionally, we further identified its availability in rapeseed (Brassica napus L.) and sesame (Sesamum indicum). High polymorphism percent (75%) of the designed primers indicated it is a general method and can be applied in other species.
The method provided in this study can generate primers more effectively for every SNP site compared to other AS-PCR primer design methods. The high allele-specific efficiency of the SNP primer allows the feasibility for low- to moderate- throughput SNP analyses and is much suitable for gene mapping, map-based cloning, and marker-assisted selection in crops.
- Gut IG: Automation in genotyping of single nucleotide polymorphisms. Hum Mutat 2001, 17:475–492. CrossRef
- Kwok PY: Methods for genotyping single nucleotide polymorphisms. Annu Rev Genomics Hum Genet 2001, 2:235–258. CrossRef
- Flint-Garcia SA, Thornsberry JM, Buckler ES: Structure of linkage disequilibrium in plants. Annu Rev Plant Biol 2003, 54:357–374. CrossRef
- Wang C, Liu Z: Arabidopsis ribonucleotidereductases are critical for cell cycle progression, DNA damage repair, and plant development. Plant Cell 2006, 18:350–365. CrossRef
- Feltus FA, Wan J, Schulze SR, Estill JC, Jiang N, Paterson AH: An SNP resource for rice genetics and breeding based on subspecies indica and japonica genome alignments. Genome Res 2004, 14:1812–1819. CrossRef
- Hillier LW, Miller RD, Baird SE, Chinwalla A, Fulton LA, Koboldt DC, Waterston RH: Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny. PLoS Biol 2007, 5:e167. CrossRef
- Eberle MA, Ng PC, Kuhn K, Zhou L, Peiffer DA, Galver L, Viaud-Martinez KA, Lawley CT, Gunderson KL, Shen R, Murray SS: Power to detect risk alleles using genome-wide tag SNP panels. PLoS Genet 2007, 3:1827–1837. CrossRef
- Shendure J, Mitra RD, Varma C, Church GM: Advanced sequencing technologies: methods and goals. Nat Rev Genet 2004, 5:335–344. CrossRef
- Barbazuk WB, Emrich SJ, Chen HD, Li L, Schnable PS: SNP discovery via 454 transcriptome sequencing. Plant J 2007, 51:910–918. CrossRef
- Trick M, Long Y, Meng J, Bancroft I: Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencing. Plant Biotechnol J 2009, 7:334–346. CrossRef
- Li R, Li Y, Fang X, Yang H, Wang J, Kristiansen K, Wang J: SNP detection for massively parallel whole-genome resequencing. Genome Res 2009, 19:1124–1132. CrossRef
- Livak KJ, Marmaro J, Todd JA: Towards fully automated genome-wide polymorphism screening. Nat Genet 1995, 9:341–342. CrossRef
- Myakishev MV, Khripin Y, Hu S, Hamer DH: High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genome Res 2001, 11:163–169. CrossRef
- Huang J, Wei W, Zhang J, Liu G, Bignell GR, Stratton MR, Futreal PA, Wooster R, Jones KW, Shapero MH: Whole genome DNA copy number changes identified by high density oligonucleotide arrays. Hum Genomics 2004, 1:287–299.
- Shapero MH, Zhang J, Loraine A, Liu W, Di X, Liu G, Jones KW: MARA: a novel approach for highly multiplexed locus-specific SNP genotyping using high-density DNA oligonucleotide arrays. Nucleic Acids Res 2004, 32:e181. CrossRef
- Matsuzaki H, Dong S, Loi H, Di X, Liu G, Hubbell E, Law J, Berntsen T, Chadha M, Hui H, Yang G, Kennedy GC, Webster TA, Cawley S, Walsh PS, Jones KW, Fodor SP, Mei R: Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods 2004, 1:109–111. CrossRef
- Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Wickham Garcia E, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A: High-throughput SNP genotyping on universal bead arrays. Mutat Res 2005, 573:70–82. CrossRef
- Konieczny A, Ausubel FM: A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 1993, 4:403–410. CrossRef
- Neff MM, Turk E, Kalishman M: Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 2002, 18:613–615. CrossRef
- Thiel T, Kota R, Grosse I, Stein N, Graner A: SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development. Nucleic Acids Res 2004, 32:e5. CrossRef
- Cha RS, Zarbl H, Keohavong P, Thilly WG: Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl 1992, 2:14–20. CrossRef
- Kwok S, Chang SY, Sninsky JJ, Wang A: A guide to the design and use of mismatched and degenerate primers. PCR Methods Appl 1994, 3:S39-S47. CrossRef
- Drenkard E, Richter BG, Rozen S, Stutius LM, Angell NA, Mindrinos M, Cho RJ, Oefner PJ, Davis RW, Ausubel FM: A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiol 2000, 124:1483–1492. CrossRef
- Hayashi K, Hashimoto N, Daigen M, Ashikawa I: Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theor Appl Genet 2004, 108:1212–1220. CrossRef
- Hirotsu N, Murakami N, Kashiwagi T, Ujiie K, Ishimaru K: Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species. Plant Methods 2010, 6:12. CrossRef
- Wangkumhang P, Chaichoompu K, Ngamphiw C, Ruangrit U, Chanprasert J, Assawamakin A, Tongsima S: WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations. BMC Genomics 2007, 8:275. CrossRef
- Bui M, Liu Z: Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping. Plant Methods 2009, 5:1. CrossRef
- Tsuchihashi Z, Dracopoli NC: Progress in high throughput SNP genotyping methods. Pharmacogenomics J 2002, 2:103–110. CrossRef
- Little S: Amplification-refractory mutation system (ARMS) analysis of point mutations. Curr Protoc Hum Genet 2001, 9:9.8.1–9.8.12.
- Hua W, Li RJ, Zhan GM, Liu J, Li J, Wang XF, Liu GH, Wang HZ: Maternal control of seed oil content in Brassica napus: the role of silique wall photosynthesis. Plant J 2012, 69:432–444. CrossRef
- Li R, Li Y, Kristiansen K, Wang J: SOAP: short oligonucleotide alignment program. Bioinformatics 2008, 24:713–714. CrossRef
- An improved allele-specific PCR primer design method for SNP marker analysis and its application
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
- Online Date
- August 2012
- Online ISSN
- BioMed Central
- Additional Links
- Author Affiliations
- 1. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, People’s Republic of China
- 2. Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China