Universal Restriction Site-Free Cloning Method Using Chimeric Primers

Background: The C677T polymorphism of MTHFR gene is a risk factor of many diseases. This study is aimed at: (1) Improving a PCR-RFLP process with the own designed primers to identify the C677T polymorphism of MTHFR gene. (2) Evaluating the prevalence of the C677T polymorphism of MTHFR gene in volunteer group. Materials and method: DNA samples was extracted from peripheral blood of 60 volunteers. Designing primers by using FastPCR software, then improving PCR technique. Standardizing the optimal conditions of restriction digest by HinfI. Confirming the results of polymorphism by DNA sequencing technique. Results: We designed successfully primers to amplify fragment of MTHFR gene including C677T polymorphism and an obligatory restriction site of HinfI (as internal control). 0.5 µl of HinfI enzyme (10 U/µl) is enough for restriction digest. The MTHFR genotype frequencies were: 71.67 % (677CC); 25% (677CT); and 3.33 % (677TT). Conclusion: We standardized successfully PCR-RFLP technique to identifying C677T polymorphism of MTHFR gene. Keywords: C677T polymorphism, MTHFR gene, PCR-RFLP

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Faculty Opinions recommendation of A ligation-independent cloning method using nicking DNA endonuclease.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.6792956.6962054 ◽

2010 ◽

Author(s):

Scott Silverman

Keyword(s):

Dna Endonuclease ◽

Ligation Independent Cloning ◽

Cloning Method

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Selection on X-linked Genes During Speciation in the Drosophila athabasca Complex

Genetics ◽

10.1093/genetics/144.2.689 ◽

1996 ◽

Vol 144 (2) ◽

pp. 689-703 ◽

Cited By ~ 1

Author(s):

Michael J Ford ◽

Charles F Aquadro

Keyword(s):

Computer Simulation ◽

Natural Selection ◽

Molecular Evolution ◽

Restriction Site ◽

Neutral Model ◽

Background Selection ◽

Selective Sweeps ◽

Differential Migration ◽

Site Survey ◽

Low Levels

Abstract We present the results of a restriction site survey of variation at five loci in Drosophila athabasca, complimenting a previous study of the period locus. There is considerably greater differentiation between the three semispecies of D. athabasca at the period locus and two other X-linked genes (neon-transient-A and E74A) than at three autosomal genes (Xdh, Adh and RC98). Using a modification of the HKA test, which uses fixed differences between the semispecies and a test based on differences in Fst among loci, we show that the greater differentiation of the X-linked loci compared with the autosomal loci is inconsistent with a neutral model of molecular evolution. We explore several evolutionary scenarios by computer simulation, including differential migration of X and autosomal genes, very low levels of migration among the semispecies, selective sweeps, and background selection, and conclude that X-linked selective sweeps in at least two of the semispecies are the best explanation for the data. This evidence that natural selection acted on the X-chromosome suggests that another X-linked trait, mating song differences among the semispecies, may have been the target of selection.

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Hierarchical Analysis of Nucleotide Diversity in Geographically Structured Populations

Genetics ◽

10.1093/genetics/142.2.629 ◽

1996 ◽

Vol 142 (2) ◽

pp. 629-639 ◽

Cited By ~ 4

Author(s):

Kent E Holsinger ◽

Roberta J Mason-Gamer

Keyword(s):

Nucleotide Diversity ◽

Restriction Site ◽

Rooted Tree ◽

Structured Populations ◽

Hierarchical Analysis ◽

Restriction Site Variation ◽

Hierarchical Levels ◽

Site Variation ◽

The Mean ◽

Mean Time

Abstract Existing methods for analyzing nucleotide diversity require investigators to identify relevant hierarchical levels before beginning the analysis. We describe a method that partitions diversity into hierarchical components while allowing any structure present in the data to emerge naturally. We present an unbiased version of Nei's nucleotide diversity statistics and show that our modification has the same properties as Wright's F ST. We compare its statistical properties with several other F ST estimators, and we describe how to use these statistics to produce a rooted tree of relationships among the sampled populations in which the mean time to coalescence of haplotypes drawn from populations belonging to the same node is smaller than the mean time to coalescence of haplotypes drawn from populations belonging to different nodes. We illustrate the method by applying it to data from a recent survey of restriction site variation in the chloroplast genome of Coreopsis grandiflora.

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Identification and Target-Modification of SL-BBI: A Novel Bowman–Birk Type Trypsin Inhibitor from Sylvirana latouchii

Biomolecules ◽

10.3390/biom10091254 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1254 ◽

Cited By ~ 1

Author(s):

Xi Chen ◽

Dong Chen ◽

Linyuan Huang ◽

Xiaoling Chen ◽

Mei Zhou ◽

...

Keyword(s):

Antimicrobial Activity ◽

Trypsin Inhibitor ◽

Inhibitory Activity ◽

Membrane Damage ◽

Antiproliferative Effects ◽

Docking Simulations ◽

Cell Membrane Damage ◽

Action Mode ◽

Trypsin Inhibition ◽

Cloning Method

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

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