Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics Article Swipe
A. Abdul Kader Jailani
,
Anirudha Chattopadhyay
,
P. Lava Kumar
,
Oinam Washington Singh
,
Sunil Kumar Mukherjee
,
Anirban Roy
,
Neeti Sanan‐Mishra
,
Bikash Mandal
·
YOU?
·
· 2023
· Open Access
·
· DOI: https://doi.org/10.3390/v15122332
YOU?
·
· 2023
· Open Access
·
· DOI: https://doi.org/10.3390/v15122332
Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed ‘long-fragment circular-efficient PCR’ (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable ‘all in one’ technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.
Related Topics
Concepts
Biology
Functional genomics
Plasmid
Genome
Nicotiana benthamiana
Genetics
Capsid
Gene
Restriction digest
Computational biology
Insert (composites)
Multiple cloning site
Restriction enzyme
Virology
Expression vector
Genomics
Recombinant DNA
Engineering
Mechanical engineering
Metadata
- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.3390/v15122332
- https://www.mdpi.com/1999-4915/15/12/2332/pdf?version=1701183127
- OA Status
- gold
- Cited By
- 4
- References
- 51
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4389099948
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
-
https://openalex.org/W4389099948Canonical identifier for this work in OpenAlex
- DOI
-
https://doi.org/10.3390/v15122332Digital Object Identifier
- Title
-
Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional GenomicsWork title
- Type
-
articleOpenAlex work type
- Language
-
enPrimary language
- Publication year
-
2023Year of publication
- Publication date
-
2023-11-28Full publication date if available
- Authors
-
A. Abdul Kader Jailani, Anirudha Chattopadhyay, P. Lava Kumar, Oinam Washington Singh, Sunil Kumar Mukherjee, Anirban Roy, Neeti Sanan‐Mishra, Bikash MandalList of authors in order
- Landing page
-
https://doi.org/10.3390/v15122332Publisher landing page
- PDF URL
-
https://www.mdpi.com/1999-4915/15/12/2332/pdf?version=1701183127Direct link to full text PDF
- Open access
-
YesWhether a free full text is available
- OA status
-
goldOpen access status per OpenAlex
- OA URL
-
https://www.mdpi.com/1999-4915/15/12/2332/pdf?version=1701183127Direct OA link when available
- Concepts
-
Biology, Functional genomics, Plasmid, Genome, Nicotiana benthamiana, Genetics, Capsid, Gene, Restriction digest, Computational biology, Insert (composites), Multiple cloning site, Restriction enzyme, Virology, Expression vector, Genomics, Recombinant DNA, Engineering, Mechanical engineeringTop concepts (fields/topics) attached by OpenAlex
- Cited by
-
4Total citation count in OpenAlex
- Citations by year (recent)
-
2025: 1, 2024: 3Per-year citation counts (last 5 years)
- References (count)
-
51Number of works referenced by this work
- Related works (count)
-
10Other works algorithmically related by OpenAlex
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| abstract_inverted_index.two | 70 |
| abstract_inverted_index.was | 107, 161 |
| abstract_inverted_index.(210 | 115 |
| abstract_inverted_index.(770 | 131 |
| abstract_inverted_index.16.4 | 144 |
| abstract_inverted_index.This | 65, 92, 160 |
| abstract_inverted_index.acid | 204 |
| abstract_inverted_index.also | 244 |
| abstract_inverted_index.both | 81 |
| abstract_inverted_index.curl | 138 |
| abstract_inverted_index.done | 162 |
| abstract_inverted_index.each | 100 |
| abstract_inverted_index.ends | 82 |
| abstract_inverted_index.from | 122 |
| abstract_inverted_index.full | 127 |
| abstract_inverted_index.gene | 121, 130, 167, 255, 305 |
| abstract_inverted_index.into | 142 |
| abstract_inverted_index.lack | 23 |
| abstract_inverted_index.leaf | 137 |
| abstract_inverted_index.need | 312 |
| abstract_inverted_index.only | 236 |
| abstract_inverted_index.part | 277 |
| abstract_inverted_index.that | 79 |
| abstract_inverted_index.this | 58 |
| abstract_inverted_index.wide | 248 |
| abstract_inverted_index.with | 74, 96 |
| abstract_inverted_index.CGMMV | 207 |
| abstract_inverted_index.Delhi | 140 |
| abstract_inverted_index.Thus, | 286 |
| abstract_inverted_index.amino | 203 |
| abstract_inverted_index.could | 185 |
| abstract_inverted_index.days. | 285 |
| abstract_inverted_index.green | 152 |
| abstract_inverted_index.human | 254 |
| abstract_inverted_index.kits, | 269 |
| abstract_inverted_index.large | 41, 193, 300 |
| abstract_inverted_index.newly | 85, 101 |
| abstract_inverted_index.novel | 54 |
| abstract_inverted_index.often | 28 |
| abstract_inverted_index.plant | 240 |
| abstract_inverted_index.quick | 271 |
| abstract_inverted_index.range | 249 |
| abstract_inverted_index.sites | 27 |
| abstract_inverted_index.their | 217 |
| abstract_inverted_index.these | 49, 214 |
| abstract_inverted_index.under | 257 |
| abstract_inverted_index.viral | 241 |
| abstract_inverted_index.virus | 155 |
| abstract_inverted_index.(VIGS) | 170 |
| abstract_inverted_index.LC-PCR | 105, 184, 225, 233, 262, 288 |
| abstract_inverted_index.PCR’ | 63 |
| abstract_inverted_index.assays | 212 |
| abstract_inverted_index.capsid | 128 |
| abstract_inverted_index.cloned | 157 |
| abstract_inverted_index.couple | 283 |
| abstract_inverted_index.formed | 102 |
| abstract_inverted_index.insert | 111 |
| abstract_inverted_index.mosaic | 154 |
| abstract_inverted_index.mottle | 153 |
| abstract_inverted_index.one’ | 296 |
| abstract_inverted_index.plant, | 251 |
| abstract_inverted_index.planta | 211 |
| abstract_inverted_index.primer | 78 |
| abstract_inverted_index.proves | 289 |
| abstract_inverted_index.region | 194 |
| abstract_inverted_index.relies | 14 |
| abstract_inverted_index.rounds | 71 |
| abstract_inverted_index.single | 202 |
| abstract_inverted_index.strand | 87 |
| abstract_inverted_index.study, | 59 |
| abstract_inverted_index.termed | 60 |
| abstract_inverted_index.unique | 25 |
| abstract_inverted_index.vector | 169, 174 |
| abstract_inverted_index.virus) | 141 |
| abstract_inverted_index.within | 281 |
| abstract_inverted_index.‘all | 294 |
| abstract_inverted_index.(tomato | 136 |
| abstract_inverted_index.Various | 209 |
| abstract_inverted_index.address | 48 |
| abstract_inverted_index.animal, | 252 |
| abstract_inverted_index.applied | 187 |
| abstract_inverted_index.crucial | 3 |
| abstract_inverted_index.develop | 164 |
| abstract_inverted_index.enzyme) | 196 |
| abstract_inverted_index.foreign | 177 |
| abstract_inverted_index.genome. | 208 |
| abstract_inverted_index.genomes | 242 |
| abstract_inverted_index.genomic | 12 |
| abstract_inverted_index.hinders | 29 |
| abstract_inverted_index.partial | 113 |
| abstract_inverted_index.plants, | 180 |
| abstract_inverted_index.plasmid | 7, 42, 90, 301 |
| abstract_inverted_index.present | 52 |
| abstract_inverted_index.protein | 129, 178 |
| abstract_inverted_index.results | 93 |
| abstract_inverted_index.strand. | 103 |
| abstract_inverted_index.through | 303 |
| abstract_inverted_index.utility | 222 |
| abstract_inverted_index.(CGMMV), | 156 |
| abstract_inverted_index.However, | 21 |
| abstract_inverted_index.cloning, | 1 |
| abstract_inverted_index.cucumber | 151 |
| abstract_inverted_index.deletion | 190 |
| abstract_inverted_index.employed | 109 |
| abstract_inverted_index.enabling | 270 |
| abstract_inverted_index.in-vitro | 258 |
| abstract_inverted_index.intended | 9 |
| abstract_inverted_index.involves | 67 |
| abstract_inverted_index.ligation | 19, 39 |
| abstract_inverted_index.multiple | 33 |
| abstract_inverted_index.nick-gap | 98 |
| abstract_inverted_index.pCambia. | 159 |
| abstract_inverted_index.phytoene | 119 |
| abstract_inverted_index.provides | 264 |
| abstract_inverted_index.sequence | 114 |
| abstract_inverted_index.strategy | 56 |
| abstract_inverted_index.studies, | 13 |
| abstract_inverted_index.suitable | 237, 293 |
| abstract_inverted_index.validate | 216 |
| abstract_inverted_index.(LC-PCR). | 64 |
| abstract_inverted_index.Molecular | 0 |
| abstract_inverted_index.Moreover, | 35 |
| abstract_inverted_index.Nicotiana | 123 |
| abstract_inverted_index.achieving | 36 |
| abstract_inverted_index.construct | 30, 147 |
| abstract_inverted_index.deletion, | 307 |
| abstract_inverted_index.expensive | 268 |
| abstract_inverted_index.genomics. | 231 |
| abstract_inverted_index.ligation. | 316 |
| abstract_inverted_index.modifying | 239, 299 |
| abstract_inverted_index.mutation, | 309 |
| abstract_inverted_index.silencing | 168 |
| abstract_inverted_index.technique | 66, 106, 226, 263, 297 |
| abstract_inverted_index.(replicase | 195 |
| abstract_inverted_index.additional | 76 |
| abstract_inverted_index.applicable | 245 |
| abstract_inverted_index.biological | 218 |
| abstract_inverted_index.construct. | 91 |
| abstract_inverted_index.constructs | 8, 43, 215, 302 |
| abstract_inverted_index.desaturase | 120 |
| abstract_inverted_index.expression | 173 |
| abstract_inverted_index.frequently | 45 |
| abstract_inverted_index.functional | 11, 230 |
| abstract_inverted_index.infectious | 146 |
| abstract_inverted_index.insertion, | 306 |
| abstract_inverted_index.nucleotide | 280 |
| abstract_inverted_index.processes. | 20 |
| abstract_inverted_index.successful | 16, 38 |
| abstract_inverted_index.third-long | 77 |
| abstract_inverted_index.alternative | 266 |
| abstract_inverted_index.begomovirus | 135 |
| abstract_inverted_index.benthamiana | 124 |
| abstract_inverted_index.complements | 80 |
| abstract_inverted_index.conditions. | 259 |
| abstract_inverted_index.deciphering | 228 |
| abstract_inverted_index.eliminating | 310 |
| abstract_inverted_index.engineering | 6, 256 |
| abstract_inverted_index.plant-virus | 229 |
| abstract_inverted_index.restriction | 17, 26, 314 |
| abstract_inverted_index.synthesized | 86 |
| abstract_inverted_index.Furthermore, | 182 |
| abstract_inverted_index.challenging. | 46 |
| abstract_inverted_index.highlighting | 220 |
| abstract_inverted_index.introduction | 272 |
| abstract_inverted_index.limitations, | 50 |
| abstract_inverted_index.nucleotides) | 116, 132 |
| abstract_inverted_index.preparation, | 31 |
| abstract_inverted_index.prerequisite | 4 |
| abstract_inverted_index.substitution | 199 |
| abstract_inverted_index.successfully | 108 |
| abstract_inverted_index.tobamovirus, | 150 |
| abstract_inverted_index.Additionally, | 260 |
| abstract_inverted_index.modifications | 274 |
| abstract_inverted_index.necessitating | 32 |
| abstract_inverted_index.respectively. | 181 |
| abstract_inverted_index.site-directed | 304 |
| abstract_inverted_index.virus-induced | 166 |
| abstract_inverted_index.functionality, | 219 |
| abstract_inverted_index.modifications. | 34 |
| abstract_inverted_index.‘long-fragment | 61 |
| abstract_inverted_index.circular-efficient | 62 |
| abstract_inverted_index.self-circularization | 95 |
| cited_by_percentile_year.max | 97 |
| cited_by_percentile_year.min | 91 |
| corresponding_author_ids | https://openalex.org/A5108739061, https://openalex.org/A5102810336 |
| countries_distinct_count | 2 |
| institutions_distinct_count | 8 |
| corresponding_institution_ids | https://openalex.org/I11874761, https://openalex.org/I33213144, https://openalex.org/I4210142124, https://openalex.org/I45509622 |
| citation_normalized_percentile.value | 0.88116368 |
| citation_normalized_percentile.is_in_top_1_percent | False |
| citation_normalized_percentile.is_in_top_10_percent | False |