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Related Concept Videos

Genome Annotation and Assembly03:36

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The genome refers to all of the genetic material in an organism. It can range from a few million base pairs in microbial cells to several billion base pairs in many eukaryotic organisms. Genome assembly refers to the process of taking the DNA sequencing data and putting it all back together in a correct order to create a close representation of the original genome. This is followed by the identification of functional elements on the newly assembled genome, a process called genome annotation.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly
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AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

Fanli Zeng1, Jinping Zang1, Suhua Zhang2

  • 1College of Life Sciences, Hebei Agricultural University, Baoding, 071001, People's Republic of China.

BMC Biotechnology
|November 16, 2017
PubMed
Summary
This summary is machine-generated.

We developed AFEAP cloning, a scarless DNA assembly method for synthetic biology. This powerful tool efficiently constructs large DNA molecules and biological circuits from multiple fragments.

Keywords:
Assembly of fragment ends after PCRBacterial artificial chromosomesDNA assemblyMulti-fragment assemblySynthetic biology

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biotechnology

Background:

  • Advances in DNA assembly are crucial for synthetic biology.
  • Existing tools may not meet the demands of complex genetic constructions.
  • New methods are needed for modular and reliable DNA assembly.

Purpose of the Study:

  • To introduce AFEAP cloning, a novel DNA assembly method.
  • To demonstrate its capability for scarless and modular construction of genetic elements.
  • To provide a new tool for synthetic biologists.

Main Methods:

  • AFEAP cloning involves two PCR rounds followed by sticky-end ligation.
  • A second asymmetric PCR introduces overlapping overhangs for fragment annealing.
  • T4 DNA ligase seals nicks, and bacterial transformation yields plasmids.

Main Results:

  • AFEAP cloning achieved high accuracy (80-100%) assembling 1-13 fragments into an 8 kb plasmid.
  • Large plasmids (up to 35.6 kb) were assembled from five fragments with over 80% fidelity.
  • Construction of a 200 kb bacterial artificial chromosome (BAC) was demonstrated with 46.7% fidelity.

Conclusions:

  • AFEAP cloning is a powerful, efficient, and sequence-independent DNA assembly tool.
  • It enables the assembly of numerous fragments (up to 13) and large DNA constructs (up to 200 kb).
  • This method expands the toolkit available for synthetic biology applications.