<commit_before>---
layout: page
title: Barcoding
---

What is barcoding?
==================

At the supermarket, the cashier scans the barcode of your groceries. The widths
of the black stripes on the produces are read by a scanner and converted into a
number. This number is unique to each grocery so it can be looked up in a
database to determine what it is and its price.

In the early 2000s, a group of scientists from the University of Guelph in
Ontario, Canada, proposed to apply a similar approach to life. They thought that
by using DNA, it should be possible to find a portion of DNA that would be
unique to each species, and therefore that could be used to identify species the
same way a barcode identify groceries at the supermarket.

In animal cells, DNA can be found in the nucleus and in the
mitochondria. Different parts of the DNA evolve at different rates. Some evolve
fast and can be used to distinguish between individuals of the same
species. These are the parts that are used in forensic tests to identify crime
suspects. Other parts evolve really slowly such that the same DNA sequence is
very similar from bacteria to humans. In between these two extremes, a portion
of the mitochondrial genome, the cytochrome oxidase I (COI) has been identified
to evolve at the appropriate rate to be able to differentiate between species
across most animals. This gene is now considered the “barcoding gene”, and
typically about 650 base pairs (the DNA letters) are sequenced.

Protein coding genes such as COI evolve by mutations: base pairs composing the
DNA sequence (A, C, T and G) are substituted by another base pair. Chance and
natural selection interplay, and after multiple generations of interbreeding,
these new mutations will be shared among individuals of the same species.

One of the strengths of barcoding compared to morphologically-based
identification methods is that because it is based on genetics, it tracks the
interbreeding individuals, and therefore match closely our modern species
concept (i.e., “a group of living organisms consisting of individuals capable of
interbreeding and producing fertile offsprings”). However, barcoding is not
without any issues, and can be misleading in some (rare) situations.

How do you obtain barcodes?
===========================

Obtaining the barcode from a animal involves three steps:

- DNA extraction: to separate the DNA from the rest of the cell component and
    makes it available for amplification;
- Barcode amplification: to generate many copies of the barcode so it can be
    sequenced;
- Barcode sequencing: to convert the DNA molecule into a series of letters that
    can be analyzed and be used to identify the organisms from which it’s from.

DNA extraction
--------------

DNA stores the information that the cells use to function. Because of this
critical role, DNA is protected within the cells by the double membrane of the
nucleus or the mitochondrion. Furthermore, because DNA is a long and thin
molecule, it is wrapped around proteins. Therefore, we first need to isolate the
DNA. In other words, we need to make it accessible by breaking down the cell
membranes and dissociate it from the proteins it is attached to.

To this end, a cocktail of chemical products are used to break down the cell
membranes and the proteins: chaotropic salts that destabilize chemical bonds,
detergents to solubilize lipids from the cell membranes and proteins, and
enzymes (mostly proteinases) to denature the proteins. To separate these cell
debris and the DNA, most modern DNA extraction protocols involve a silica
membrane that binds DNA molecules but let the cell debris go through. To remove
any other potential chemical compounds (e.g., sugars, pigments), the membrane is
washed several times. Finally, the membrane is dried and the DNA is released by
eluting it in a buffer with a slightly basic pH to keep the DNA molecule stable
for several years when stored in a freezer.

Here, we will use a faster protocol that doesn’t generate a pure DNA solution
but works very well for barcoding purposes with most organisms. It uses a
combination of heat, proteinases and a chelating ligand called Chelex.

### How to do it?: Protocol for the Chelex Extraction

1.  Sterilize your forceps and scissors by flaming them over an alcohol
    burner. This will remove any contaminants from your instruments.

2.  Cut a small piece of tissue about the size of this dot \( \bullet \) (1
    mm<sup>2</sup> from your sample and place it in a 2 mL tube. Larger tissue
    samples may inhibit your PCR reactions.

3.  Add 150 µL of Chelex bead solution to your 2 mL tube.

4.  Add 10 µL of Proteinase K to your tube.

5.  Spin your tube in the centrifuge for several seconds. Make sure the
    centrifuge is balanced with every tube having a similar tube opposite it.

6.  Incubate your tube at 95°C for at least 20 minutes.

Barcode amplification
---------------------

Once the DNA is isolated, we want to amplify the barcode. The amplification
process produces millions of copies of the barcode. The process of generating
these copies is called PCR (Polymerase Chain Reaction). It uses a mix of
chemical products and enzymes to amplify a specific part of the DNA. This
cocktail needs to include:

- the template: a small amount of your DNA solution that was extracted from your
    sample;

- the primers: these two short fragments of DNA of known sequences will anneal
    (i.e., attach) to the DNA from your sample (template) and target the
    fragment of DNA you wish to amplify;

- the oligonucleotides: a solution of the building blocks of DNA molecules, the
    single letters (A, C, T, G) that will be assembled to create the copies of
    the barcodes;

- the polymerase: this enzyme will assemble the oligonucleotides to create the<commit_msg>attempt to use bootstrap panel<commit_after>---
layout: page
title: Barcoding
---

What is barcoding?
==================

At the supermarket, the cashier scans the barcode of your groceries. The widths
of the black stripes on the produces are read by a scanner and converted into a
number. This number is unique to each grocery so it can be looked up in a
database to determine what it is and its price.

In the early 2000s, a group of scientists from the University of Guelph in
Ontario, Canada, proposed to apply a similar approach to life. They thought that
by using DNA, it should be possible to find a portion of DNA that would be
unique to each species, and therefore that could be used to identify species the
same way a barcode identify groceries at the supermarket.

In animal cells, DNA can be found in the nucleus and in the
mitochondria. Different parts of the DNA evolve at different rates. Some evolve
fast and can be used to distinguish between individuals of the same
species. These are the parts that are used in forensic tests to identify crime
suspects. Other parts evolve really slowly such that the same DNA sequence is
very similar from bacteria to humans. In between these two extremes, a portion
of the mitochondrial genome, the cytochrome oxidase I (COI) has been identified
to evolve at the appropriate rate to be able to differentiate between species
across most animals. This gene is now considered the “barcoding gene”, and
typically about 650 base pairs (the DNA letters) are sequenced.

Protein coding genes such as COI evolve by mutations: base pairs composing the
DNA sequence (A, C, T and G) are substituted by another base pair. Chance and
natural selection interplay, and after multiple generations of interbreeding,
these new mutations will be shared among individuals of the same species.

One of the strengths of barcoding compared to morphologically-based
identification methods is that because it is based on genetics, it tracks the
interbreeding individuals, and therefore match closely our modern species
concept (i.e., “a group of living organisms consisting of individuals capable of
interbreeding and producing fertile offsprings”). However, barcoding is not
without any issues, and can be misleading in some (rare) situations.

How do you obtain barcodes?
===========================

Obtaining the barcode from a animal involves three steps:

- DNA extraction: to separate the DNA from the rest of the cell component and
    makes it available for amplification;
- Barcode amplification: to generate many copies of the barcode so it can be
    sequenced;
- Barcode sequencing: to convert the DNA molecule into a series of letters that
    can be analyzed and be used to identify the organisms from which it’s from.

DNA extraction
--------------

DNA stores the information that the cells use to function. Because of this
critical role, DNA is protected within the cells by the double membrane of the
nucleus or the mitochondrion. Furthermore, because DNA is a long and thin
molecule, it is wrapped around proteins. Therefore, we first need to isolate the
DNA. In other words, we need to make it accessible by breaking down the cell
membranes and dissociate it from the proteins it is attached to.

To this end, a cocktail of chemical products are used to break down the cell
membranes and the proteins: chaotropic salts that destabilize chemical bonds,
detergents to solubilize lipids from the cell membranes and proteins, and
enzymes (mostly proteinases) to denature the proteins. To separate these cell
debris and the DNA, most modern DNA extraction protocols involve a silica
membrane that binds DNA molecules but let the cell debris go through. To remove
any other potential chemical compounds (e.g., sugars, pigments), the membrane is
washed several times. Finally, the membrane is dried and the DNA is released by
eluting it in a buffer with a slightly basic pH to keep the DNA molecule stable
for several years when stored in a freezer.

Here, we will use a faster protocol that doesn’t generate a pure DNA solution
but works very well for barcoding purposes with most organisms. It uses a
combination of heat, proteinases and a chelating ligand called Chelex.

<div class="panel panel-primary">
### How to do it?: Protocol for the Chelex Extraction

1.  Sterilize your forceps and scissors by flaming them over an alcohol
    burner. This will remove any contaminants from your instruments.

2.  Cut a small piece of tissue about the size of this dot \( \bullet \) (1
    mm<sup>2</sup> from your sample and place it in a 2 mL tube. Larger tissue
    samples may inhibit your PCR reactions.

3.  Add 150 µL of Chelex bead solution to your 2 mL tube.

4.  Add 10 µL of Proteinase K to your tube.

5.  Spin your tube in the centrifuge for several seconds. Make sure the
    centrifuge is balanced with every tube having a similar tube opposite it.

6.  Incubate your tube at 95°C for at least 20 minutes.
</div>

Barcode amplification
---------------------

Once the DNA is isolated, we want to amplify the barcode. The amplification
process produces millions of copies of the barcode. The process of generating
these copies is called PCR (Polymerase Chain Reaction). It uses a mix of
chemical products and enzymes to amplify a specific part of the DNA. This
cocktail needs to include:

- the template: a small amount of your DNA solution that was extracted from your
    sample;

- the primers: these two short fragments of DNA of known sequences will anneal
    (i.e., attach) to the DNA from your sample (template) and target the
    fragment of DNA you wish to amplify;

- the oligonucleotides: a solution of the building blocks of DNA molecules, the
    single letters (A, C, T, G) that will be assembled to create the copies of
    the barcodes;

- the polymerase: this enzyme will assemble the oligonucleotides to create the
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