mehdi behdani

Molecular deoxyribonucleic acid markers are one of the most important tools in molecular biology labs. The size of DNA molecule is determined by comparing them with known bands of markers during gel electrophoresis. In this study, we have suggested an efficient strategy to produce molecular weight markers in an industrial scale.

A combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR), was used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the bands of ladder and produce the DNA fragment by plasmid linearization through digestion. In the PCR method, the DNA fragments with length 102 bp lesser than the related bands in DNA ladder are amplified by PCR and cloned in pTZ57T/A cloning vector. Then, PCRs with forward and reverse 100‑bp primers on the resulting plasmids amplify the ladder fragments. F100 and R100 primers bind to the backbone of pTZ57R (without insert) and amplify a 100‑bp PCR product. PCR on the plasmid with insert amplifies DNA fragment with 102+ insert length bp size.

Upon application of this strategy, 2000-10,000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100-1500 bp fragments were produced during PCR using only a set of forward and reverse (100 bp) primers.

The highest advantage of this cost–benefit approach is to produce different types of molecular weight markers by using an effective and short protocol.

As we all know, sperm has the capacity to take up foreign DNA, therefore, sperm mediated gen transfer can be an inexpensive and simple method in animal transgenesis in various species. However, there is not sufficient evidence of DNA uptake by ovine spermatozoa. The purpose of the present study was to examine the uptake of human lysozyme gene contained plasmid (pEGFP-IRES-hLys) by ovin spermatozoa. In the first experiment, semen was prepared from three ram (each ram two times) by electrical method. After removal of seminal plasma, 1x106 spermatozoa were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 15, 30, 60 and 120 minutes and then observed for motility, uptake percent and uptake intensity by florescent microscopy. Also after 60 minutes incubation sperms were treated by DNaseI to assay adsorption or uptake of pEGFP-IRES-hLys. In the second experiment, washed and unwashed sperms were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 30 and 60 minutes to evaluate the effect of presence seminal plasma on sperm uptake and motility. The findings showed that increasing incubation time increased number/percentage of spermatozoa carrying exogenous DNA and its intensity. But this different was significant only up to 30 minutes. We found that 60.16% of the cells were bound to DNA after 120 minutes incubation. Incubation with exogenous DNA induced a slightly decrease in sperm total and progressive motility. But no post acrosom uptaked sperm was motile. After treatment with DNaseI, strong florescent emission from post acrosom indicated absorption of pEGFP-IRES-hLys by spermatozoa. Presence of seminal plasma induced a slightly decrease in percent of DNA absorbed spermatozoa and absorption intensity, but did not inhibit completely. Ram spermatozoa showed a high capacity to bind DNA quickly and reach a maximum after 30 min. However, no sperm with real uptake (post acrosomal) was motile. Incubation with lower DNA concentration and/or shorter time may be helpful.

The Rifampicin resistance and susceptibility of Mycobacterium tuberculosis are caused by mutations in the 81-base pair region of the rpoB gene encoding the b-subunit of RNA polymerase.

Isoniazid resistance of M. tuberculosis is related to mutations in inha , oxyR and ahpC genes which 30 to 90 percent of Isoniazid resistance is occurred in 3015 codons of katg gene. The rpoB and katG sequences of 30 isolates were analyzed to identify the mutations and compare the mutations with their related susceptibilities.

In this research, we investigated the location and type of rpoB and katG mutations in Mycobacterium tuberculosis which had been achieved from Pasteur Institute of Tehran. PCR Amplification and DNA sequencing methods were performed. In this assay, from 507 to 537 codons and 315 codons of rpoB and katG genes were sequenced and also mutations were analyzed, respectively.

Angiogenesis or new blood vessels generation is the most important factor affecting cell growth and proliferation in physiologic and pathologic conditions. Placenta Growth Factor (PLGF) is one of the important proteins for angiogenesis induction. In this study, placenta-derived PLGF-1 cDNA was cloned and expressed in Escherichia coli expression system.

In this experimental study, the human placenta-derived PLGF-1 gene was amplified using specific primers and was cloned into pET32a and pET28a expression vectors. In order to express target protein, pET32a-PLGF-1 and pET28a-PLGF-1 constructs were transformed into E.coli Rosetta. PLGF-1 expression was induced by IPTG, and then the yield of expressed protein and its solubility was analyzed by SDS-PAGE and Western blotting assay.

The pET32a-PLGF-1 and pET28a-PLGF-1 constructs were confirmed by sequencing. The bacterial lysates derived from IPTG-induced samples showed exact proteinand confirmed by western bloting. The majority of the expressed protein was insoluble.

The PLGF-1 is well expressed in Rosetta E.coli system and it could be used in different project.

Molecular DNA markers are one of the most important tools in molecular biology labs. The size of DNA molecules is determined by comparing them with known bands of markers during gel electrophoresis. There are many different protocols to produce these kinds of molecular markers. In this study we have suggested an efficient strategy to produce molecular weight markers in industrial proportions. To achieve the desired sizes of DNA fragments, a combination of two previously known methods, restriction enzyme digestion and polymerase chain reaction (PCR), were used. The enzymatic digestion process was based on designing and constructing plasmids which equaled in size with the desired length of DNA fragments and produced the desired DNA fragment upon linearization. In the PCR method, the desired length of DNA fragments were cloned in multiple cloning sites of pTZ57R plasmid and in a PCR reaction, the new constructed plasmid was used as a template to produce the final fragment. Upon application of this strategy, 2000 and 3000 bp DNA fragments were produced by enzymatic digestion of plasmids of the same size. Moreover, 100 to 1500 bp fragments were produced during PCR using only a set of forward and reverse primers at the flanking region of pTZ57R multiple cloning site. The highest advantage of this cost-benefit approach is to produce different types of molecular weight markers by using an effective and short protocol.