originally commercialized as SNaPshot for analysis of single-nucleotide polymorphisms by capillary electrophoresis (CE). Application of this general method to bisulfite-converted/PCR-amplified genomic DNA (gDNA) to analytically infer polymorphic methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich regions of gDNA has been noted previously by others to be limited by CE mobility-shifted peaks for SBE products derived from guanine (G)/ adenine (A)-mixed-base primers used to hybridize to possible polymorphic sites (i.e., C vs. thymine [T] resulting from 5mC vs. C, respectively). This limitation is precluded in the current study by using novel SNaPshot primers modified with N6-methoxy-2,6-diaminopurine (K), which was originally described in 1991 by Brown and Lin as a unique adenineguanine analog capable of participating in three H-bonds with C or T in DNA. Oligonucleotides modified by K as a bispecific complement for C/T are commercially available or can be readily synthesized, and they may have utility in other assay formats used to analyze CpG methylation status.Developing multiplexed SNP assays with special reference to degraded DNA templates.pdfAbstract: This protocol describes a single nucleotide polymorphism (SNP) genotyping strategy for highly degraded DNA, using a two-stage multiplex whereby multiple fragments are first amplified in a single exponential reaction and the products of this PCR are added to a linear single-base-extension reaction. It utilizes the analytical power of a capillary electrophoresis system to simultaneously type all the target sites. The protocol is specifically written for use with severely fragmented templates, typical of ancient DNA, and can be adapted to widely used detection platforms. The addition of the single-phase genotyping step avoids the need for the re-amplification and cloning of PCR products, while providing its own controls for the detection of contamination and allelic drop-out. This protocol can facilitate the routine analysis of up to 52 SNP markers (haploid or diploid) in 96 samples in a single day, and is recommended for the authentication of data in all areas of DNA research (population and medical genetics, forensics, ancient DNA).Identification and Genotyping of Mycobacterium tuberculosis Complex Species by Use of a SNaPshot Minisequencing-Based Assay.pdfAbstract: The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allelespecific minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped. This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely related members of the MTBC, the distinction between principal genetic groups, and the characterization of M. tuberculosis isolates into one of the seven prominent