We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.

The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

ScV is a double-stranded RNA virus of yeast consisting of two separately encapsidated dsRNAs (L and M). ScV-1 and ScV-2 are two dsRNA viruses present in two different yeast killer strains, K1 and K2. Our 3' end sequence analysis shows that the two sets of viral dsRNAs from ScV-1 and ScV-2 are very similar. Consensus sequences for transcriptase and replicase initiation are proposed. A stem and loop structure with a 3' terminal AUGC sequence, like that of several plant virus plus strand RNAs, is present at the putative replicase initiation site of one of the yeast viral RNA plus strands.

Background: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (−1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. Methods: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 −1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. Results: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. Conclusions: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2–based diagnostics have key advantages.

Previously , the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele , our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNAclamp SMAP-2) , we could detect KRAS mutations within 60 minutes , including sample preparation. We compared results from PNA-clamp SMAP-2 assay , polymerase chain reaction-restriction fragment length polymorphism , and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNAclamp SMAP-2 method is a rapid , simple , and highly sensitive detection assay for cancer mutations. (J Mol