Simultaneous splicing of multiple DNA fragments in one PCR reaction

BACKGROUND: Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments. RESULTS: We performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5–10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification. CONCLUSIONS: Our method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match

N-(9H-Fluoren-9-yl­idene)-4-methyl­aniline

In the title compound, C20H15N, the fluorene unit is essentially planar [r.m.s. deviation 0.0334 Å] and the benzene ring bound to the imine N atom bears a methyl group which is nearly coplanar [dihedral angle 0.5 (1)°]. The dihedral angle between the substituent benzene ring and the 9H-fluoren-9-imine unit is 71.1 (3)°. Inter­molecular π–π inter­actions between the benzene rings of adjacent fluorene units [centroid–centroid distance 3.8081 (13) Å] are present in the crystal structure, resulting in a one-dimensional supra­molecular architecture

Chlorido[(E)-2-hydr­oxy-6-(isonicotinoyl­hydrazonometh­yl)phen­yl]mercury(II) monohydrate

The asymmetric unit of the title compound, [Hg(C13H10N3O2)Cl]·H2O, contains two independent mercury(II) complexes with slightly different conformations, related via a pseudo-inversion centre, and two water mol­ecules. The HgII atoms show a typical linear geometry to a C atom of the benzene ring and to a Cl atom. A benzene C and the azomethine N atom chelate the HgII atoms with weak intra­molecular Hg⋯N bonding distances of 2.735 (3) and 2.739 (3) Å, respectively. The resulting five-membered metallacycles are nearly coplanar with the benzene rings [dihedral angles = 0.9 (1) and 0.7 (1)°], while the pyridine rings make dihedral angles with the benzene units of 58.17 (1) and 56.58 (1)°. In the crystal structure, the HgII complexes are linked by hydr­oxy donor and pyridine acceptor groups into chains along [010]. The water mol­ecules connect the complexes through inter­molecular O—H⋯Ocarbon­yl bonds in the a-axis direction, and the azomethine H atoms donate towards the water O atoms, forming a three-dimensional network of inter­molecular O—H⋯N, O—H⋯O and N—H⋯O hydrogen bonds

Long-Term Follow-Up of the Fellow Eye in Patients Undergoing Surgery on One Eye for Treating Myopic Traction Maculopathy

Objective. To observe the fellow eye in patients undergoing surgery on one eye for treating myopic traction maculopathy. Methods. 99 fellow eyes of consecutive patients who underwent unilateral surgery to treat MTM were retrospectively evaluated. All patients underwent thorough ophthalmologic examinations, including age, gender, duration of follow-up, refraction, axial length, intraocular pressure, lens status, presence/absence of a staphyloma, and best-corrected visual acuity (BCVA). Fundus photographs and SD-OCT images were obtained. When feasible, MP-1 microperimetry was performed to evaluate macular sensitivity and fixation stability. Results. At an average follow-up time of 24.7 months, 7% fellow eyes exhibited partial or complete MTM resolution, 68% stabilized, and 25% exhibited progression of MTM. Of the 38 eyes with “normal” macular structure on initial examination, 11% exhibited disease progression. The difference in progression rates in Groups 2, 3, and 4 was statistically significant. Refraction, axial length, the frequency of a posterior staphyloma, chorioretinal atrophy, initial BCVA, final BCVA, and retinal sensitivity all differed significantly among Groups 1–4. Conclusions. Long axial length, chorioretinal atrophy, a posterior staphyloma, and anterior traction contribute to MTM development. Patients with high myopia and unilateral MTM require regular OCT monitoring of the fellow eye to assess progression to myopic pre-MTM. For cases exhibiting one or more potential risk factors, early surgical intervention may maximize the visual outcomes

Roles of TNF-α gene polymorphisms in the occurrence and progress of SARS-Cov infection: A case-control study

<p>Abstract</p> <p>Background</p> <p>Host genetic factors may play a role in the occurrence and progress of SARS-Cov infection. This study was to investigate the relationship between tumor necrosis factor (TNF)-<it>α </it>gene polymorphisms with the occurrence of SARS-CoV infection and its role in prognosis of patients with lung interstitial fibrosis and femoral head osteonecrosis.</p> <p>Methods</p> <p>The association between genetic polymorphisms of <it>TNF-α </it>gene and susceptibility to severe acute respiratory syndromes (SARS) was conducted in a hospital-based case-control study including 75 SARS patients, 41 health care workers and 92 healthy controls. Relationships of TNF-α gene polymorphisms with interstitial lung fibrosis and femoral head osteonecrosis were carried out in two case-case studies in discharged SARS patients. PCR sequencing based typing (PCR-SBT) method was used to determine the polymorphisms of <it>TNF-α </it>gene in locus of the promoter region and univariate logistic analysis was conducted in analyzing the collected data.</p> <p>Results</p> <p>Compared to TT genotype, the CT genotype at the -204 locus was found associated with a protective effect on SARS with OR(95%<it>CI</it>) of 0.95(0.90–0.99). Also, TT genotype, CT and CC were found associated with a risk effect on femoral head necrosis with ORs(95%<it>CI</it>) of 5.33(1.39–20.45) and 5.67(2.74–11.71), respectively and the glucocorticoid adjusted OR of CT was 5.25(95%CI 1.18–23.46) and the combined (CT and CC) genotype OR was 6.0 (95%<it>CI </it>1.60–22.55) at -1031 site of <it>TNF-α </it>gene. At the same time, the -863 AC genotype was manifested as another risk effect associated with femoral head necrosis with OR(95%<it>CI</it>) of 6.42(1.53–26.88) and the adjusted OR was 8.40(95%CI 1.76–40.02) in cured SARS patients compared to CC genotype.</p> <p>Conclusion</p> <p>SNPs of <it>TNF-α </it>gene of promoter region may not associate with SARS-CoV infection. And these SNPs may not affect interstitial lung fibrosis in cured SARS patients. However, the -1031CT/CC and -863 AC genotypes may be risk factors of femoral head necrosis in discharged SARS patients.</p