A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.

[1]  W. Thilly,et al.  Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. , 1992, PCR methods and applications.

[2]  M. Daly,et al.  MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. , 1987, Genomics.

[3]  U Hammerling,et al.  Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. , 2000, Genome research.

[4]  N. Arnheim,et al.  Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[5]  R. Amasino,et al.  A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. , 1998, The Plant journal : for cell and molecular biology.

[6]  J Chory,et al.  dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. , 1998, The Plant journal : for cell and molecular biology.

[7]  K. Livak,et al.  Allelic discrimination using fluorogenic probes and the 5' nuclease assay. , 1999, Genetic analysis : biomolecular engineering.

[8]  B. K. Pal,et al.  Allele specific polymerase chain reaction , 1991 .

[9]  C. Levenson,et al.  Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. , 1990, Nucleic acids research.

[10]  F. Ausubel,et al.  A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. , 1993, The Plant journal : for cell and molecular biology.

[11]  M Ronaghi,et al.  Direct analysis of single‐nucleotide polymorphism on double‐stranded DNA by pyrosequencing , 2000, Biotechnology and applied biochemistry.

[12]  K. Edwards,et al.  A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. , 1991, Nucleic acids research.

[13]  K. Livak,et al.  A homogeneous, ligase-mediated DNA diagnostic test. , 1998, Genome research.

[14]  I. Tinoco,et al.  Comparison between DNA melting thermodynamics and DNA polymerase fidelity. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[15]  N. Arnheim,et al.  Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. , 1992, Nucleic acids research.

[16]  C. Lister,et al.  Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana , 1993 .

[17]  C Summers,et al.  Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). , 1989, Nucleic acids research.

[18]  S. Kwok,et al.  A guide to the design and use of mismatched and degenerate primers. , 1994, PCR methods and applications.

[19]  M. Boosalis,et al.  Nearest neighbor influences on DNA polymerase insertion fidelity. , 1989, The Journal of biological chemistry.

[20]  M. Uhlén,et al.  Single-nucleotide polymorphism analysis by pyrosequencing. , 2000, Analytical biochemistry.

[21]  Earl Hubbell,et al.  Genome-wide mapping with biallelic markers in Arabidopsis thaliana , 1999, Nature Genetics.

[22]  J. Ecker,et al.  Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. , 1994, Genomics.