Minisequencing

Suomalainen A, Syvanen AC. Analysis of nucleotide sequence variations by solid-phase minisequencing. Methods Mol Biol 2003 226 361-366. 

Liljedahl U, Karlsson J, Melhus H, Kurland L, Lindersson M, Kahan T, Nystrom F, Lind L, Syvanen AC. A microarray minisequencing system for pharmacogenetic profiling of antihypertensive drug response. Pharmacogenetics 2003 13 7-17. 

Multiplex Minisequencing on Microarrays Application to Pharmacogenetics of Antihypertensive Drug Response... 

In minisequencing single nucleotide primer extension (SNE) or single base extension (SBE), a DNA polymerase is used to extend a detection primer, which anneals immediately adjacent to the site of the SNP, with a labeled nucleotide analog (23,24). In the microarray format of... 

Figure 1 In the array-of-arrays conformation a standard microscope slide is divided into 80 subarrays with a diameter identical to that of a 384-well-microtiter-plate reaction well, schematically illustrated to the left in the image. Up to 200 oligonucleotide spots can be printed per subarray at a center-to-center distance of 200 pm. With the possibility to analyze 80 samples in parallel, up to 16,000 genotypes per slide can be generated. Fluorescence images of four reaction wells, where the minisequencing reaction have been performed are shown to the right.

Figure 2 Minisequencing reaction on with extension primers immobilized on the microarray surface. The multiplex PCR products of the regions containing the SNPs are allowed to hybridize to the oligonucleotides immobilized on the microarray (A). The primers are extended with fluorescently labelled ddNTPs complementary to the nucleotide at the SNP position. Four different fluorophores, one for each nucleotide is used allowing for a simultaneous detection of the four nucleotides in one single reaction (B). After washing the slide with sodium hydroxide and salt buffers, only the extended primers covalently attached to the surface remains and the fluorescence is measured (C) and the genotypes assigned.

Syvanen AC. From gels to chips minisequencing primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum Mutat 1999 13 1-10. 

Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvanen AC. Minisequencing a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 1997 7 606-614. 

Raitio M, Lindroos K, Laukkanen M, Pastinen T, Sistonen P, Sajantila A, Syvanen AC. Y-chromosomal SNPs in Finno-Ugric-speaking populations analyzed by minisequencing on microarrays. Genome Res 2001 11 471 482. 

Lindroos K, Liljedahl U, Raitio M, Syvanen AC. Minisequencing on oligonucleotide microarrays comparison of immobilisation chemistries. Nucleic Acids Res 2001 29 e69. 

Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, Syvanen AC. Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res 2003 31 el29. 

Liljedahl U, Fredriksson M, Dahlgren A, Syvanen A-C. Detecting imbalanced expression of SNP alleles by minisequencing on microarrays. BMC Biotechnol 2004 4 24. 

Schwartz M, Sorensen N, Hansen FJ, Hertz JM, Norby S, Tranebjaerg L, Skovby F. Quantification, by the solid-phase minisequencing, of the telomeric and centromeric copies of the survival motor neuron gene in families with spinal muscular atrophy. Hum Mol Genet 1997 6(1) 99 104. 

Sitbon, G. Syvanen, A.-C. Multiplex fluorescent minisequencing applied to the typing of genes encoding... 

Wang W, Kham SKY, Yeo G-H, Quah T-C, Chong SS. Multiplex minisequencing screen for common Southeast Asian and Indian p thalassemia mutations. Clin Chem 2003 49 209-213. 

Wang W, Ma ESK, Chan AYY, Chui DHK, Chong.SS. Multiple minisequencing screen for seven Southeast Asian non-deletional a-thalassemia mutations. Clin Chem 2003 49 800-3. 

Single Nucleotide Extension Assay Also known as single-base primer extension or minisequencing, single nucleotide extension (SNE) assays involve the... 

Two alternative methods can be used for SNP analysis using microarrays. The first is based on differential hybridization (31) and the other on minisequencing (32). For differential hybridization, a set of oligonucleotides (containing the SNP of interest) is arrayed onto a glass slide. The set com-... 

Alternatively, oligonucleotides are arrayed onto the glass slide and a one-base minisequencing reaction is carried out on the slide with the arrayed oligonuleotides as the primer and sample PCR products as the template. SNP is typed by scanning the array to determine which fluorescent dideoxynucleotide has been incorporated. Again many SNPs can be typed at once by minisequencing from a multiplex of PCR products. 

Nyren, P., Pettersson, B., Uhlen, M. 1993. Solid phase DNA minisequencing by enzymatic luminometric inorganic pyrophosphate detection assay. Anal. Biochem. 208 171-175. 

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