Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

Riet, Job van; Ramos, Laia; Lewis, Randolph V.; Marins, Luís Fernando

doi:10.4238/gmr16039796

Cited by 15 publications

(13 citation statements)

References 15 publications

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“…The process was included denaturation at 94°C for 20 Sec., then Annealing at 65°C for 2 min. 15 • Preparation of mixture 2 (M2): Four µl of (Mix1) was added to another tube containing 2 µl of 100 mmol/L NEB buffer containing 5mmol/L (CH 3 COO) 2 Mg and 1.6 µl of Klenow DNA pol. And 4 µl of one substrate of either (α-dATP-s, dTTP, dGTP, dCTP) mixed and incubated for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Impact of Anti-Toxoplasma gondii and adipose hormones with Insulin Resistant on obese aborted women

Hassan¹,

Mohammed²,

Shweash³

et al. 2020

ijddt

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This study was carried out at Baghdad hospital in, for the period from November 2018 to July 2019. The study included (151) aborted obese women whose ages ranged between (18–41) years with positive Toxoplasma gondii infection. They were divided into two groups according to the body mass index (BMI) value: Group 1: consisted of 61 women with BMI less than 30, Group 2: consisted of (90) women with BMI greater than 30. The control group included (52) healthy volunteer women aged 19–41 years with negative Toxoplasma for comparison of the results. The case and controls were matched for age and gender. Serum samples were tested for fasting blood sugar, insulin, IgG, and IgM of Toxoplasma, Leptin, and Adiponectin as well as insulin resistance index. The results showed that the age factor was not significant between group 1 and group 2 when compared with the healthy group, and there was no significant change between group 2 comparing to group1. In this study, the result of BMI showed substantial increase in group 1, while highly marked increase in group 2 when both groups were compared with the control group. Finally, the levels of Toxoplasma IgG and IgM antibodies showed a highly significant increase in the two patient groups in comparison with the control group. An increase in mean value of leptin concentration was noticeable in group 1 and group 2 with a highly significant difference when compared with the control group. No significant difference was found in the levels of fasting blood glucose in Group 1 and Group 2 compared to the control group. Also, a significant difference in HOMA-IR and QUICK- IR was observed in the patient groups once associated control group. Data revealed a considerable difference with the glucose/insulin ratio in group 1, but a highly significant was noticed in group 2 when compared with the control group. HOMA-AD results showed a significant difference in Group 1 and a highly significant decrease in Group 2.

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Section: Methodsmentioning

confidence: 99%

Impact of Anti-Toxoplasma gondii and adipose hormones with Insulin Resistant on obese aborted women

Hassan¹,

Mohammed²,

Shweash³

et al. 2020

ijddt

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“…The GAANTRY system may minimize the number of transformations, the number of antibiotics needed for the selection, and could reduce the subsequent plant breeding steps. The cloning strategies recently developed for long repeat sequences (Riet et al, 2017) and the recursive directional ligation approach (Dinjaski et al, 2018) may be used to create donor plasmids for the GAANTRY system. Combinations of different sequence modules from the donor plasmid will essentially allow an array of molecules, each with a unique combination of domains.…”

Section: Gaantry Systemmentioning

confidence: 99%

Advances in Plant-Derived Scaffold Proteins

2020

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Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for regenerative medicine and drug screening. The increasing demand for such applications urges solutions for cost effective and sustainable supplies of hypoallergenic and biocompatible scaffold proteins. Here, we summarize recent efforts in obtaining plantderived biosynthetic spider silk analogue and the extracellular matrix protein, collagen. Both proteins are composed of a large number of tandem block repeats, which makes production in bacterial hosts challenging. Furthermore, post-translational modification of collagen is essential for its function which requires co-transformation of multiple copies of human prolyl 4-hydroxylase. We discuss our perspectives on how the GAANTRY system could potentially assist the production of native-sized spider dragline silk proteins and prolyl hydroxylated collagen. The potential of recombinant scaffold proteins in drug delivery and drug discovery is also addressed.

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“…(2) Some vectors are difficult to amplify by PCR. For example, the GC content of the vector is too high or too low, or the vector contains too many repetitive sequences, which will affect PCR and even render the reaction impossible to continue [ 29 – 31 ]. (3) As previously mentioned, if vectors cannot be linearized by PCR or fidelity is vital for the experiment, the methods shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly

Fan

et al. 2021

BMC Biotechnol

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Background The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes. Results We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated. Conclusions Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.

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Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

Cited by 15 publications

References 15 publications

Impact of Anti-Toxoplasma gondii and adipose hormones with Insulin Resistant on obese aborted women

Impact of Anti-Toxoplasma gondii and adipose hormones with Insulin Resistant on obese aborted women

Advances in Plant-Derived Scaffold Proteins

Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly

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