Tail variation of the folding primer affects the SmartAmp2 process differently

Folding primer (FP), together with turn-back primer (TP) and boost primer (BP), is one of the major components of SmartAmp2, a rapid amplification-based method for SNP detection. FP has a unique design where the annealing region is combined with a tail that can fold back. FP tails can be classified as either “strong” or “weak”, depending on the melting temperature and free energy of the hairpin structure. We report that FP tails affect the amplification process differently; by changing the FP concentration, we can increase the amplification reaction speed with “strong tails”. Unlike “strong tails”, concentration change of FP with “weak tails” did not show significant impact on the amplification speed. The comparative analyses using gel electrophoresis demonstrate that the FP type and FP ratio in the reaction change the amplification pattern. The above observations can be used to optimize the reaction and manipulate the reaction speed of SmartAmp2.     

Rapid Detection of SNP (c.309T>G) in the MDM2 Gene by the Duplex SmartAmp Method

Background

Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans.

Methodology

In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named “Duplex SmartAmp,” which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms.

Results and Conclusions

By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.     

Normalization of Epigenetic Change in the Genome by Peptide Bioregulator (Ala–Glu–Asp–Gly) in Pulmonary Tuberculosis

International Journal of Peptide Research and Therapeutics - The aim of this study was to evaluate genetic and epigenetic variation of the genome in patients with sensitive pulmonary tuberculosis...     

Effects of the turn-back primer on intermediate product generation in isothermal DNA amplification

In DNA amplification, the initial step of copying a target sequence from the template DNA--the so-called intermediate product generation step--is very important. In examining the turn-back primer (TP)-dependent isothermal DNA amplification (TIA) method, we determined the actual time point of intermediate product generation by extrapolating dsDNA amplification curves. Our results indicate that intermediate product creation is the rate-limiting step in TIA, and good TP design is advantageous for improving the intermediate production process.     

A large inversion in the linear chromosome of <Emphasis Type="Italic">Streptomyces griseus</Emphasis> caused by replicative transposition of a new Tn<Emphasis Type="Italic">3</Emphasis> family transposon

We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.     

High-depth sequencing of over 750 genes supports linear progression of primary tumors and metastases in most patients with liver-limited metastatic colorectal cancer

BackgroundColorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC.ResultsHigh depth Illumina sequencing and use of three different variant callers enable comprehensive and accurate identification of somatic variants down to 2.5% variant allele frequency. We identify a median of 11 somatic single nucleotide variants (SNVs) per tumor. Across patients, a median of 79.3% of somatic SNVs present in the primary are present in the metastasis and 81.7% of all alterations present in the metastasis are present in the primary. Private alterations are found at lower allele frequencies; a different mutational signature characterized shared and private variants, suggesting distinct mutational processes. Using B-allele frequencies of heterozygous germline SNPs and copy number profiling, we find that broad regions of allelic imbalance and focal copy number changes, respectively, are generally shared between the primary tumor and metastasis.ConclusionsOur analyses point to high genomic concordance of primary tumor and metastasis, with a thick common trunk and smaller genomic branches in general support of the linear progression model in most patients with liver-limited mCRC. More extensive studies are warranted to further characterize genomic progression in this important clinical population.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0589-1) contains supplementary material, which is available to authorized users.     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ⁻ mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome. Received: 29 July 1996 / Accepted: 27 August 1996     

The tap‐tpg gene pair on the linear plasmid functions to maintain a linear topology of the chromosome in Streptomyces rochei

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2‐L (211 kb), pSLA2‐M (113 kb) and pSLA2‐S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2‐L and pSLA2‐M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2‐39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2‐L from strain 51252, which carries only pSLA2‐L, also resulted in terminal deletions in newly obtained mutants. The tap‐tpg gene pair, which encodes a telomere‐associated protein and a terminal protein for end patching, is located on pSLA2‐L and pSLA2–M but has not hitherto been found on the chromosome. These results led us to the idea that the tap‐tpg of pSLA2‐L or pSLA2‐M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap‐tpg of pSLA2‐M.