PCR cloning of partial <i>nbs</i> sequences from grape (<i>Vitis aestivalis</i> Michx)

Chang, Ming‐Mei; DiGennaro, Peter; Macula, Anthony J.

doi:10.1002/bmb.20320

Cited by 3 publications

(10 citation statements)

References 33 publications

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“…A ~4,270 bp DNA fragment and a ~500 bp DNA insert were observed in Eco RI and Eco RI + Pst I double digest lanes (Figure 2a). The ~500 bp bands are similar in size to those of previously cloned grape nbs sequences 21,25 . A ~4800 bp DNA fragment was seen in the Pst I digested lane, indicating a single Pst I site in the plasmid.…”

Section: Resultssupporting

confidence: 67%

“…To ensure the success of the lab exercise carried out by undergraduates who had little or no experience with molecular techniques, a 4‐week lab module on “plasmid‐to‐plasmid” Southern blot analysis was implemented to validate the presence of nbs sequences in their cloned plasmids from a preceding lab module like those described in the previous paper 21 …”

Section: Introductionmentioning

confidence: 99%

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Plasmid‐to‐plasmid Southern blot analysis validates the presence of nucleotide binding site (nbs) sequences in cloned plasmids

Chang

2022

Biochem Molecular Bio Educ

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Southern blot analysis is an important molecular biology technique for identifying a specific sequence in DNA samples. Although it is no longer used extensively in recent years, the steps and underlying principles of Southern blot are applicable to modern biology. High sensitivity and limited background are keys to successful Southern blots, whereas obtaining good quality and quantity of genomic DNA as starting materials and detecting a single/low copy target sequence in the genome can be challenging. To ensure student success in performing the technique for the first time, a modified "plasmid-to-plasmid" Southern blot was implemented to confirm the presence of grape nucleotidebinding site (nbs) sequences in cloned plasmids like those described previously. The plasmid DNA and a control plasmid, pSCA7 (T1-T3-W6) containing a known grape nbs sequence, were digested with restriction enzymes, followed by agarose gel electrophoresis. The DNA band corresponding to the nbs sequence of the pSCA7 (T1-T3-W6) was extracted from the gel for PCR digoxigenin (DIG) probe synthesis. At the same time, the cloned plasmid DNA and its digested DNA fragments were blotted from the gel onto nylon membranes to be hybridized with the DIG probe followed by the detection for nbs sequences. Students successfully performed Southern blots to confirm the presence of nbs sequences in their cloned plasmids and wrote up the results following the format of scientific research papers. They learned the principles and applications of Southern blot and gained hands-on experience with associated techniques.

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Section: Resultssupporting

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Plasmid‐to‐plasmid Southern blot analysis validates the presence of nucleotide binding site (nbs) sequences in cloned plasmids

Chang

2022

Biochem Molecular Bio Educ

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“…Candidate genes can also be targeted for full gene sequence isolation. The methodology and detailed protocols for these downstream techniques have been previously reported [6].…”

Section: Discussionmentioning

confidence: 99%

“…Hence, students may often face difficulties in understanding fundamental concepts of protein beyond the commonly used "Locks and Keys" concept [5]. Deeper understanding and a better appreciation of the concepts involved in many protein-protein interactions can be achieved by providing a hands-on experience [6] and relating it to actual research applications.…”

Section: Introductionmentioning

confidence: 99%

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise

Yeo

Foong

Tam

et al. 2017

Biochem Molecular Bio Educ

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Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding siteleucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcriptionpolymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins.

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“…The integrity and concentration of DNA samples were verified by electrophoresis on 0.8% agarose gel stained with ethidium bromide and by spectrophotometry. To identify polymorphisms related to pathogen resistance in Psidium species, RGA markers were used, which derived from degenerate primers of the P-loop motif from the domains NBS (S2 and F1), GLPLAL (As1, As2, As3, and LM637), and RNBS-B (R1) (Kanazin et al, 1996;Leister et al, 1996;Yu et al, 1996;Chang et al, 2009) (Table S1).…”

Section: Molecular Analysismentioning

confidence: 99%

Relationship between Psidium species (Myrtaceae) by resistance gene analog markers: focus on nematode resistance

et al. 2017

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Guava (Psidium guajava L.) crop is severely affected by the nematode Meloidogyne enterolobii. Native Psidium species have been reported as sources of resistance against this nematode. Knowledge on the molecular relationship between Psidium species based on plant resistance gene analogs (RGA) can be useful in the genetic breeding of guava for resistance to M. enterolobii. In this study, RGA markers from conserved domains, and structural features of plant R genes, were employed to characterize Psidium species and establish genetic proximity, with a focus on nematode resistance. SSR markers were also applied owing to their neutral nature, thus differing from RGA markers. For this, species reported as sources of resistance to M. enterolobii, such as P. cattleianum and P. friedrichsthalianum, as well as species occurring in the Atlantic Rainforest and susceptible genotypes, were investigated. In 10 evaluated Psidium species, high interspecific genetic variability was verified through RGA and SSR markers, with intraspecific variation in P. guajava higher with SSR, as was expected. Resistant species were clustered by RGA markers, and differential amplicons among genotypes resistant and susceptible to M. enterolobii were identified. Knowledge on the molecular relationships between Psidium species constitutes useful information for breeding of the guava tree, providing direction for hybridization and material for rootstocks. Additionally, the genetic relationship between native species, which have been little studied, and P. guajava were estimated by RGAs, which were confirmed as important markers for genetic diversity related to pathogen resistance.

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PCR cloning of partial nbs sequences from grape (Vitis aestivalis Michx)

Cited by 3 publications

References 33 publications

Plasmid‐to‐plasmid Southern blot analysis validates the presence of nucleotide binding site (nbs) sequences in cloned plasmids

Plasmid‐to‐plasmid Southern blot analysis validates the presence of nucleotide binding site (nbs) sequences in cloned plasmids

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise

Relationship between Psidium species (Myrtaceae) by resistance gene analog markers: focus on nematode resistance

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