A Simple and Easy Method for Site-specific Mutagenesis Using Long-distance Inverse PCR in the Presence of Pfu-DNA Polymerase

Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.

Isolation of the Flanking Sequences Adjacent to Transgenic T-DNA in Brassica napus Genome by an Improved Inverse PCR Method

[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...

Rapid Amplification of 5′ cDNA End of S. Liaotungensis Choline Monooxygenase Using Inverse PCR RACE

Based on part of a known cDNA sequence of Suaeda Liaotungensis choline monooxygenase, the authors successfully cloned the 5′ cDNA end of Suaeda Lianotungensis choline monooxygenase using Inverse PCR RACE with a specially designed 5′-phosphated RT primer and two pairs of specific inverse PCR primers. Compared with the anchored PCR RACE, inverse PCR RACE has better specificity and higher amplification.

Clone and Bioinformatics Analysis of Pig SRPK1 Gene 5' UTR Region

Part 5＇ UTR region of pig SRPK1 gene was cloned by inverse PCR （I-PCR）, then a 425 bp gene sequence was acquired. A promoter region in -1--309 bp was predicted by online tool （TFSEARCH） and 32 binding sites of transcription with scores higher than 85 were getten, of which scores of Spl, MyoD, and HSF2 were over 90. Some of these binding sites of transcription factors were connected with promoters, but TATA-box, which was important to gene expression, hadn＇t been found in this region. By using PCR-SSCP method to search SNPs this part （5＇ UTR of SRPK1 in pig）, total of 40 Large White pigs were obtained as the research objects, but no polymorphism were found. Thus, 5＇ UTR of SRPK1 was speculated with a characteristic of high conservation, while it might have been directly or indirectly selected in commercial breeding. The paper provided a further feature of SRPK1 gene in molecular genetics.

Potential dissemination mechanism of the tetC gene in Aeromonas media from the aerobic biofilm reactor under oxytetracycline stresses

The tet C gene has been found to be one of the most widely distributed tetracycline resistance( tet) genes in various environmental niches, but the detailed dissemination mechanisms are still largely unknown. In the present study, 11 tet C-containing Aeromonas media strains were isolated from an aerobic biofilm reactor under oxytetracycline stresses, and the genome of one strain was sequenced using the Pac Bio RSII sequencing approach to reveal the genetic environment of tet C. The tet C gene was carried by an IS 26 composite transposon, named Tn 6434. The tet C-carrying Tn 6434 structure was detected in all of the A. media strains either in a novel plasmid p Aeme2( n = 9) or other DNA molecules( n = 2) by PCR screening. The NCBI database searching result shows that this structure was also present in the plasmids or chromosomes of other 13 genera, indicating the transferability of Tn 6434. Inverse PCR and sequencing confirmed that Tn 6434 can form a circular intermediate and is able to incorporate into a preexisting IS 26 element, suggesting that Tn 6434 might be responsible for the dissemination of tet C between different DNA molecules. This study will be helpful in uncovering the spread mechanism of tet genes in water environments.