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PLASMID IDENTIFICATION USING DUALLY LABELLED DNA BARCODES

Torstensson, Erik LU (2020) FYTM04 20192
Computational Biology and Biological Physics - Has been reorganised
Abstract
DNA barcoding is a powerful and inexpensive tool for identifying genetic material. Identification of plasmids is especially important for tracking the global spread of anti-biotic resistance. In this study, new methods were presented for identifying plasmids using DNA barcodes that combine two very different DNA labelling types. Labelling using the affinity based technique of competitive binding was combined with sequence specific cuts made by restriction enzymes. DNA barcodes using two label types were matched to a database of reference sequences and were shown to have a better matching performance compared to using only one label type. Two plasmids of lengths 130 kilo-base-pairs and 220 kilo-base-pairs respectively were experimentally... (More)
DNA barcoding is a powerful and inexpensive tool for identifying genetic material. Identification of plasmids is especially important for tracking the global spread of anti-biotic resistance. In this study, new methods were presented for identifying plasmids using DNA barcodes that combine two very different DNA labelling types. Labelling using the affinity based technique of competitive binding was combined with sequence specific cuts made by restriction enzymes. DNA barcodes using two label types were matched to a database of reference sequences and were shown to have a better matching performance compared to using only one label type. Two plasmids of lengths 130 kilo-base-pairs and 220 kilo-base-pairs respectively were experimentally measured in nanofluidic channels and successfully matched to reference sequences. In order to demonstrate proof of concept for the identification procedure, emulated experiments were performed for every reference sequence in the NCBI RefSeq database of fully sequenced plasmids. For plasmids ≥ 100 kbp, DNA barcodes based on competitive binding identified the correct plasmid 93% of the time, barcodes based on restriction enzyme cutting identified the correct plasmid 57% of the time, and dually labelled barcodes identified the correct plasmid 96% of the time. For shorter plasmids 20 − 100 kbp, DNA barcodes based on competitive binding identified the correct plasmid 55% of the time, barcodes based on restriction enzyme cutting identified the correct plasmid 17% of the time, and dually labelled barcodes identified the correct plasmid 59% of the time. Finally, we have shown how re-sampling statistics can be used for a significant increase in the proportion of uniquely identified plasmids. For plasmids ≥ 100 kbp, the proportion of unique matches increased from 38% to 54% for DNA barcodes based on competitive binding, from 32% to 35% for barcodes based on restriction enzyme cutting, and from 48% to 59% for dually labelled barcodes. For shorter plasmids 20−100 kbp there was a negligible increase in the proportion of unique matches. (Less)
Popular Abstract
Like the rungs of a ladder, the base-pairs of the DNA molecule form a very thin chain with a vast contour length unlike any other molecule.
Hidden in plain sight within the base-pair sequence is the genetic code which is the cell's recipe for synthesizing proteins, the building blocks of all life as we know it.

To have the ability of quickly reading and interpreting DNA has always been a pipe dream within the field of bioinformatics, and the search for a figurative magnifying glass for DNA has driven us to solutions that could be described as deceptively simple.
New advancements in nano-technology have raised enthusiasm for methods that do not require large-scale culturing of cells or replication of DNA, and with streamlined... (More)
Like the rungs of a ladder, the base-pairs of the DNA molecule form a very thin chain with a vast contour length unlike any other molecule.
Hidden in plain sight within the base-pair sequence is the genetic code which is the cell's recipe for synthesizing proteins, the building blocks of all life as we know it.

To have the ability of quickly reading and interpreting DNA has always been a pipe dream within the field of bioinformatics, and the search for a figurative magnifying glass for DNA has driven us to solutions that could be described as deceptively simple.
New advancements in nano-technology have raised enthusiasm for methods that do not require large-scale culturing of cells or replication of DNA, and with streamlined fabrication of nanofluidic systems this could come at a bargain price.
A nanofluidic chip uses delicate channels, like a sieve for the molecular realm, to coerce DNA into stretching and unravelling its secrets.

By staining the molecule with a sequence-specific fluorescent dye, the method of optical DNA mapping aims to capture the complexity of the overarching structure on an imaging device.
Bearing in mind some inherent constraints on imaging resolution, the pattern obtained from optically mapping a genome tends to be many orders of magnitude less complex than the underlying base-pair sequence.
In spite of this lower complexity, the DNA barcode contains enough information to be recognizable when compared to a reference genome, a literal barcode for inventorying the produce of life.

The new angle of attack in this study is to combine the fluorescent pattern obtained from optical DNA mapping with a molecular label created by an enzyme that binds to a specific combination of base-pairs.
This technology could lead way to new innovative approaches in tackling problems such as tracking the spread of genes culpable for anti-biotic resistance, or identifying bacteria by their genome as a diagnostics tool in a clinical setting. (Less)
Please use this url to cite or link to this publication:
author
Torstensson, Erik LU
supervisor
organization
course
FYTM04 20192
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9005516
date added to LUP
2020-03-02 14:41:14
date last changed
2020-03-02 14:41:14
@misc{9005516,
  abstract     = {{DNA barcoding is a powerful and inexpensive tool for identifying genetic material. Identification of plasmids is especially important for tracking the global spread of anti-biotic resistance. In this study, new methods were presented for identifying plasmids using DNA barcodes that combine two very different DNA labelling types. Labelling using the affinity based technique of competitive binding was combined with sequence specific cuts made by restriction enzymes. DNA barcodes using two label types were matched to a database of reference sequences and were shown to have a better matching performance compared to using only one label type. Two plasmids of lengths 130 kilo-base-pairs and 220 kilo-base-pairs respectively were experimentally measured in nanofluidic channels and successfully matched to reference sequences. In order to demonstrate proof of concept for the identification procedure, emulated experiments were performed for every reference sequence in the NCBI RefSeq database of fully sequenced plasmids. For plasmids ≥ 100 kbp, DNA barcodes based on competitive binding identified the correct plasmid 93% of the time, barcodes based on restriction enzyme cutting identified the correct plasmid 57% of the time, and dually labelled barcodes identified the correct plasmid 96% of the time. For shorter plasmids 20 − 100 kbp, DNA barcodes based on competitive binding identified the correct plasmid 55% of the time, barcodes based on restriction enzyme cutting identified the correct plasmid 17% of the time, and dually labelled barcodes identified the correct plasmid 59% of the time. Finally, we have shown how re-sampling statistics can be used for a significant increase in the proportion of uniquely identified plasmids. For plasmids ≥ 100 kbp, the proportion of unique matches increased from 38% to 54% for DNA barcodes based on competitive binding, from 32% to 35% for barcodes based on restriction enzyme cutting, and from 48% to 59% for dually labelled barcodes. For shorter plasmids 20−100 kbp there was a negligible increase in the proportion of unique matches.}},
  author       = {{Torstensson, Erik}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{PLASMID IDENTIFICATION USING DUALLY LABELLED DNA BARCODES}},
  year         = {{2020}},
}