DNA You Say? Burn Everything to Burn Longer: A Guide to Leaving No Traces

No Trace Project > Risorse > DNA You Say? Burn Everything to Burn Longer: A Guide to Leaving No Traces

Note from the No Trace Project:

We hope that this translation will help English-speaking anarchists to better understand and protect themselves against the dangers of DNA.

We added many footnotes, either to add information or to explain our disagreements with the original text, especially in the section "A protocol for two". Our footnotes are preceded by “N.T.P. note”, whereas the original footnotes are not.

We did not include the original appendix which listed French forensic police laboratories and their suppliers, because we thought it wouldn't be relevant to an international audience.

Introduction

Why this publication?

DNA evidence is now widely used by the judicial system as a tool of repression. In this context, circulating unverified information such as “acetone destroys DNA” puts people at risk of repression. The aim of this zine is both to understand how the cops identify a person using DNA and to propose ways to protect oneself from identification.

We would like to see the disappearance of the prisons, the courts, the police stations and all those who participate in the machinery of repression. The idea here is to participate in building a security culture around genetic profiling. We will mainly talk about DNA, which is only one issue among many others when it comes to security in the face of repression.

DNA evidence is an article of scientific faith. No matter how much we believe in science, judges lock people up on this basis. We will use a scientific point of view, which is a partial point of view. In these pages, we do not propose a critical reflection on the use of scientific techniques around DNA identification, nor on science in general. And yet, there is a lot to think about.

What's in there?

This is a practical zine, but we'll take (brief) detours through theory. First, we'll talk about how cops work with DNA. Then we'll offer ideas for how to make their job as difficult as possible, both by giving tips to develop your own security protocols and by proposing a protocol that we consider appropriate. At the end, we have added more theoretical appendices on the biology of DNA and on some technical aspects of its use by scientists.

There are sections that use scientific jargon. The zine contains a glossary, for all words followed by an asterisk*. In the text there are numbered references [in square brackets] which are collected in the bibliography.

What we are hoping for from this zine

What we don't want is for you to take us at our word. What we want is for this zine to be modified, reappropriated, and freely distributed. There are several things that are important to us: that the text is accessible, that there is access to scientific references and so to tools for free access to science, that there are several levels of reading so that you can pick out what interests you, and especially that you feel free to modify, print, reproduce, or share it and lots of other fun stuff.

For our references, we used some books and scientific articles, which are all in the bibliography. Some of the books can be found on Libgen (Library Genesis)^[1], and some of the articles are available on Sci-Hub^[2]. These sites are accessible via the Tor Browser, although they may be occasionally down. Some scientific books are also available in university biology libraries.

Further reading

This text does not cover everything, and there are a lot of great texts on the subject, such as:

• L’apparence de la certitude : l’ADN comme “preuve” scientifique et judiciaire [The appearance of certainty: DNA as scientific and legal “proof”].
• Du sang, de la chique et du molard ! Sur l’ADN [Blood, Spit, and Snot! On DNA.].
• “Ouvrez la bouche”, dit le policier [“Open your mouth”, says the policeman].

Once upon a time, there was DNA

A living organism (like you) is made up of cells* (A on the following drawing), like the cells that make up your skin. These cells themselves have sub-compartments (e.g. B and C), which are specialized (in the production of energy, in the export of molecules…). One of these compartments is the nucleus* (C). Its role is to protect the cell's genetic information. The medium in which this information is stored is a molecule* called DNA (D) for deoxyribonucleic acid. Another compartment of the cell is called the mitochondrion* (B), it contains a little bit of DNA too. There are between 0 and 2000 mitochondria in a human cell.

DNA carries genetic information, called the genome*, that is unique to each individual (except for identical twins).

Genetic information and the genome

We can visualize DNA as a book. Inside this book, instead of clear sentences, there are sequences of letters. Most of the time, these sequences of letters do not make sense, but sometimes, in the middle, there are words that we understand. For example, we can read :

JVLKHZKHEFLKAEJLKJFASMALLBROCHURECLZAEHDFILZAH

There are sections that have no apparent meaning, which correspond to DNA sequences that are called non-coding sequences. And in the middle you can read SMALLBROCHURE, which is a piece that makes sense. This sequence would code for a trait in the cell. This is called a gene*.

A gene is a portion of DNA that carries a unit of genetic information. The majority of the human genome is made up of non-coding sequences^[3], meaning, sequences that are not genes. The genome contains all of the cell's genetic material, both coding and non-coding.

Genes can have several different forms, which are called alleles*. For example, if we imagine that there is an “eye color” gene^[4], then several different forms (alleles) of this gene will exist which will be “brown”, “blue”, “green”, “gray”, etc.

You can find more details about genes in Appendix A.

Structure of DNA

To reuse our comparison with a book, the information contained in the book is written with the 26 letters of the alphabet. DNA, on the other hand, is written with 4 letters, which are four molecular bases: A, T, C and G.

A strand of DNA is formed from four basic building blocks, the nucleotides*, called A, T, C, and G^[5]. The DNA strand is formed from a succession of these building blocks: they form a DNA sequence. The length of a DNA fragment is measured in base pairs, abbreviated as bp*. To refer to a piece of DNA, we can describe its sequence, for example ATACCACAACATCACA. If this sequence codes for properties of the cell, it is called coding DNA. The rest of the DNA is called non-coding DNA.

DNA forms a double helix from two single strands of DNA due to base pairing: A always pairs with T and C always pairs with G.

You will find more details about nucleotides in Appendix A.

This book is actually in several volumes, ranging from 1 to 23. Each volume comes in “duplicates”, that is, two versions have the same structure (for example, there will be a word on page 3, on the 4th line) but not the same content (not necessarily the same word).

Each volume represents a chromosome*. Each chromosome generally^[6] comes in two copies with one chromosome called “maternal” and one called “paternal”. For example, XX or XY for the sex chromosomes. Humans generally^[6] have 23 pairs of chromosomes. Each individual has 23 pairs of chromosome-books in each cell: we could say that an individual is a library.

The DNA double helix forms chromosomes by joining with other molecules.

Stability of the DNA molecule

Let's continue on the subject of DNA-books. The content of these books is rarely modified over time because it is protected (by the cover for example). To make this book unreadable, we try to cut it into small enough parts that we can no longer extract anything from it. For example, if we take the sequence we had before:

SMALLBROCHURE

And we cut it into many small pieces, we get the following: S, M, A, L, L, B, R, O, C, H, U, R, E. With just these letters, it is impossible to know if the base text was SMALL BROCHURE or MALL CHORE RUBS.

The cover of the book is the proteins that wrap the DNA and protect it, and the nucleus that protects everything. Cutting the DNA to make it unreadable would be equivalent to chemically attacking it, for example.

DNA is a very stable molecule, which means that it doesn't change very easily (whether from mutations or cutting the DNA strand).

All this to say that DNA is robust.

But DNA can degrade, a process through which the DNA molecule is cut into smaller and smaller fragments. Since the information is contained in how the base nucleotides are arranged in relation to each other, the smaller the sequence, the less informative it is.

There is only a single copy of nuclear DNA (contained in the nucleus) in each cell, whereas mitochondrial DNA, as a rule, exists in several hundred copies: it can therefore be analyzed in older and/or more degraded samples.

What are the police up to?

DNA collection by forensic police

First of all, it is important to specify that DNA collection is not an automatic process: sufficient quantities and a degree of quality are required for it to be usable by the cops.

Collection methods at the site

At the site of the action, the cops will use these methods to take the DNA samples to the lab for analysis:

Liquid samples (liquid blood, spit…): They are transferred to a sterile tube, refrigerated and brought to the lab as soon as possible. If the transport time is likely to be more than 24 hours, they absorb the sample with a cotton swab or sterile pad and let it dry.

Solid residue (dried blood stains on clothing…): If possible, the surface under the residue is cut out, bagged and taken away. If it cannot be cut, a sterile swab and sterile water (not too much to avoid dilution) are used to moisten and rub the sample. The cotton is dried and stored for analysis.

Touch DNA (skin cells* inside a glove, cells inside a mask around of the mouth…): The cops rub the area with slightly wet cotton or a swab and store it in a sterile tube.

Solid bits (a hair, a piece of skin that is torn off…): These are put in a sterile bag and taken away.

Usable DNA sources

This section is primarily based on Preuve par l’ADN : la génétique au service de la justice[1] [DNA evidence: genetics in the service of justice].

Blood: Blood is THE biological sample par excellence. All the DNA in blood comes from white blood cells (5,000 to 10,000 cells per μL)^[7]. Blood is very easy to analyze scientifically: in theory, any trace visible to the naked eye can be analyzed^[8].

Menstrual “blood”: Menstrual “blood” is composed of both blood and dead cells of the uterine wall. Apparently[2], this is very useful for genetic profiling.

Saliva: Saliva contains hundreds of cells from the mouth per μL. The DNA from these cells is found on licked stamps for example (a classic). A licked stamp can still be analyzed even after months or years.

Urine: Urine is not a very good source of DNA for analysis, because there is not a lot of DNA in it, and it deteriorates quickly.

Bones and teeth: DNA from bones and teeth is mainly used to identify corpses. It has a good chance of surviving, even long after death, and even if the corpse has suffered significant damage (burned, for example).

Skin samples: Intact DNA profiles can be obtained from direct skin swabs.

Objects in contact with the skin: For example, door handles, car keys, telephones, cup handles, knife handles, shoes, socks, clothing, glasses, hats, Latex gloves, pencils, watches, etc. Success rates for “Touch DNA” analysis are variable, as the amount of biological material left on surfaces varies widely among individuals (dry, clean hands leave less DNA)[3]. The DNA is often in minute quantities and sometimes degraded, so partial DNA profiles are obtained.

Note that smooth surfaces are more likely to feature fingerprints, and rough surfaces are more likely to carry DNA traces (because they scrape cells off for example).

If you touch an object with a gloved hand and then touch another object, you can contaminate the second object with the DNA that was on the first. More generally, DNA can be transferred from one object to another by contact between them[4].

Hair and body hair: The success rate of DNA analysis in the nucleus of the hair shaft cell is low (< 10%). Indeed, in the hair shaft, there is very little DNA and it is very degraded. Hair that falls out on its own has a root that is also degraded, so there are few or no living cells.

However, hair that has just fallen out or especially that has been pulled out still has its root, and nuclear DNA can be more easily analyzed. In addition, mitochondrial DNA can sometimes be used as a backup, but it is not as good as nuclear DNA for analysis. Non-human hairs can also be used and can be informative!^[9]

Cigarette butts: Another classic example: the nice cop gives you a cigarette in custody. The chances of a successful analysis are very high. Since products of combustion inhibit analysis, the paper surrounding the filter and the last millimeter of filter are carefully cut out, i.e. the parts in direct contact with the lips and mucous membranes.

Glasses, containers and food: When you drink from a glass, you deposit cells on the rim where you put your lips^[10]. This is “Touch DNA” that can be used by the cops.

Stains on fabric: If the stain had time to dry before the fabric is washed, there is a good chance that DNA is still present after washing. In the case of blood, even after machine washing at 90°C the stains can still be analyzed[5]. They are still analyzable after hand washing with soap, dish-washing liquid, or laundry detergent[6].

DNA analysis by forensic police

The basis for genetic profiling is that only twins have identical genomes*. The human genome is more or less the same in everyone, but there are differences that can be used to identify a person (genetic markers).

Genetic markers used by the cops

There are different types of variations between individuals. The ones used by the cops for identification purposes are called STRs (Short Tandem Repeats), also called microsatellites.

What is an STR?

Let's return to our DNA-book comparison and take a closer look at a sequence:

JVLKHZKHEFLFAHLHLHLHLHLHLHLAEJLKJSMALLBROCHUREFILZAH

Here's a sequence in which the pattern HL is repeated seven times. The same thing can be found in DNA, for example with repeats of the CA pattern. An STR in the above example would be the entire sequence HLHLHLHLHLHLHL, consisting of 7 repeats of the HL pattern.

In the example above, the STR is located in an area of the DNA that does not make sense (non-coding DNA), but it could also be located in the middle of a part that does make sense (coding DNA). For example, below, the STR is located right in the middle of SMALLBROCHURE:

JVLKHZKHEFLAEJLKJFASMALLBROHLHLHLHLHLHLHLHLCHUREFILZAH

The STRs analyzed by the cops are located in the non-coding regions.

The different alleles of a STR

Let's imagine that in all libraries (i.e. in all individuals), in book 4, page 32, 4th line, there is always a certain number of repetitions of the HL pattern. In some libraries, at this precise place, there are 8 repetitions of HL, while in others, there are 7, 4 or 12, for example.

In individuals, it is the same. Let's imagine a particular STR made up of repeats of the pattern GTC, located on the 3rd chromosome*, starting at 2 million base pairs from the end^[11] of the chromosome. Some individuals will have 8 repeats of the GTC pattern, while others will have 12, or 7 or whatever.

The number of repeats in a particular STR is variable. In the overall population, there may be as many as ten different versions of a particular STR, with each of the alleles* being characterized by a different number of repeats. Here, a broader definition of allele is used: the allele of a STR is the number of repetitions of the repeated pattern.

In genetic profiling, the alleles of a given number of different STRs are determined. For example, by studying different STRs numbered from 1 to 5, the number of repeats of the pattern (i.e. the allele) for each STR can be written as:

16/15(STR1); 15/7(STR2); 13/12(STR3); 5/6(STR4); 8/8(STR5)

For STR1, there are 16 repeats on one chromosome and 15 repeats on the other.

For each STR, there are two different possible numbers of repetitions, because there are two versions of each chromosome (called “maternal” and “paternal”).

Why STRs are good genetic markers

Forensic requirements for good genetic markers are as follows:

• They must be stable over a lifetime. STRs are carried by DNA, a molecule* that is fairly stable over a lifetime.
• They must be resistant to the degradation that organic structures may undergo. STRs are rather short (< 300 bp*), so have little risk of being affected by degradation. Essentially, by choosing a short sequence in a DNA molecule, there is less chance of degradation than if the sequence is longer.
• The analysis technique required must be sensitive enough to apply to minute traces. This sensitivity is what has improved the most in the last 20 years.
• The cost of the analysis must not be prohibitive. See "Costs of a DNA analysis".
• There must be enough polymorphism*, i.e. enough variation in the population, for the marker to be useful for identification. STRs have many alleles, i.e. many variants.

How is it analyzed?

The sample is first purified, meaning it is cleaned to keep only the DNA (all other substances are removed).

Then, the collected DNA is amplified, which means copied several times. This is done using PCR^[12]. PCR (Polymerase Chain Reaction) is a molecular copying technique. It is also called gene amplification. During PCR, a piece of DNA is selected and copied millions of times.

After PCR, the products are examined by gel electrophoresis^[12]. Electrophoresis is a technique that separates molecules by differences in their mobility within an electric field. For example, the more positive charges a molecule has, the more it will be attracted to the negative pole in an electric field, so the more it will move.

In gel electrophoresis, the molecules move in a gel: the larger they are, the less they move because the gel hinders their movement.

There are therefore two parameters that allow molecules to be separated in gel electrophoresis: their charge and their size. Since gel electrophoresis allows separation according to the size of the molecules, it can therefore determine the size of a piece of DNA by comparing its migration with the migration of pieces of DNA of a known size.

This technique is used for several STRs (multiplex analysis^[12]). This provides a sample's DNA profile*. Another widely used test is to determine whether the sample contains a Y chromosome.

The DNA profile that is obtained corresponds to the number of pattern repeats in a given number of STRs (15 STRs for the analysis kit called ABI used in France, for example), see the table in the section "The genetic fingerprint kept by the cops". We are not going to dwell here on the calculations that courts use to determine whether an analysis relying on a certain number of STRs is a sufficient basis to confirm an individual's identity. This is detailed in the zine L’apparence de la certitude : l’ADN comme “preuve” scientifique et judiciaire [The appearance of certainty: DNA as scientific and legal “proof”].

What influences the success of collecting the genetic material from the site?

The amount of DNA found at the site

The recommended number of cycles in PCR is 30. This allows a satisfactory DNA profile to be obtained from 0.5ng^[13] of DNA. Considering that we have about 0.006ng of DNA per cell, it takes the genetic material of about 100 cells to obtain a profile (in 2006)[1].

We will use these numbers for our calculations, but the amount of DNA needed for analysis is decreasing all the time (as we said, progress doesn't stop)[4].

There are different amounts of DNA in different sources:

• In one mL of blood, there is about 30,000 ng of DNA. It takes at least 20 millionths of a mL to do a DNA analysis that gives a satisfactory result. This is smaller than a small drop of water.
• A sample taken directly from the skin of the hand yields between 2 ng and 150 ng of DNA (on average 50 ng). Dry or recently washed skin will yield less DNA than other skin[7].
• One mL of saliva contains about 2,000 ng of DNA. Therefore, it takes at least about one millionth of a mL to do a satisfactory DNA analysis.
• One hair root yields up to 200 ng of DNA.
• In one mL of urine, 10 ng of DNA can be found.
• One mg of skin or muscle contains about 3,000 ng of DNA.
• A swab from objects held regularly by an individual yields between 1 ng and 100 ng of DNA[7].

When cops have only a few cells available, the analysis is much more vulnerable to random effects that do not reflect reality. For example, consider 3 cells containing the A and B alleles of a given gene*. When these cells undergo a degradation, the B allele does not survive. Only the A allele remains each time. The cops collect a sample and analyze it. They obtain a profile where only the A allele appears, so their interpretation will necessarily be incomplete. This is called an allele drop-out.

Even if we always leave some DNA, it is worthwhile to leave as little as possible, it is not a question of all or nothing.

Preservation conditions of the collected DNA

General considerations

DNA can be degraded before it is collected or after if it is poorly preserved by the cops who collect it[1].

In order to do analyses, they need at least a few fragments of DNA several hundred base pairs long (nucleotides*). However, several environmental factors can degrade DNA (break it):

• Some enzymes* present in the environment can cut DNA, for example enzymes produced by bacteria, or fungi present in soil. They work best at a certain temperature.
• Humidity provides one of the ingredients (water) needed to break the bonds between the nucleotides that form the DNA chain. Water is necessary for DNA degradation enzymes to act. Finally, water allows the growth of bacteria and fungi that reuse pieces of the molecules in the sample to make their own molecules (including DNA)^[14].
• Heat is one of the elements that accelerate chemical reactions that require energy, which include DNA degradation reactions.
• Light (especially UV light) chemically transforms DNA. Some skin cancers are linked to UV, for example, because UV tends to mutate DNA.
• We quote: “Of course, other aggressive chemical substances (acids, bleach…) can also attack DNA molecules. But these substances are unlikely to be encountered at crime scenes.” We'll expand on that a bit later, precisely in order to leave some at the site^[15].

Outdoors, the sun and rain leave little hope for biological traces to last beyond a few weeks to a few months. If the area where the DNA sample is left has significant microbial life (e.g. undergrowth, soil, stagnant water…), this lifespan decreases further. A hammer left in the undergrowth in the middle of summer (enzymes, humidity, heat) will keep less DNA than if it was left in winter on concrete.

Moreover, to take saliva as an example, when it is not dried, the humidity and the enzymes present rapidly degrade the DNA (in a matter of hours or days)[1].

To be properly stored, DNA must be dry, cold and protected from light. To give an idea, the recommended storage conditions for forensic samples are room temperature and low humidity for dry samples, and -20 °C for liquid samples.

A specific example: dried bloodstains

Here, we will summarize the results of an article studying the degradation of DNA in dried bloodstains[8]. It is interesting because it provides orders of magnitude of DNA degradation time. Nevertheless, these should be taken with caution, for two reasons:

• Laboratory conditions are different from those found outside.
• The DNA fragment sought by the authors is a precise sequence that is 273 bp long, whereas the STRs used by the cops are generally shorter. They therefore degrade more slowly, which would suggest that we should consider these orders of magnitude as a bare-minimum.

The results of the study are:

• Below 35°C and at less than 100% humidity, DNA degradation is negligible over the course of a year.
• For these same temperatures at 100% humidity, there is microbial growth, and various bacteria and fungi degrade the DNA. It remains analyzable for a few months (about 4 months, with large variations, for fragments of 273 bp).
• At temperatures above 45°C, no microbial growth is observed. At 45°C, the DNA remains analyzable for a long time (at least 8 to 9 months). At 55°C and 65°C, the stability of the DNA relapses, and after 3 months, the researchers cannot find the 273 bp fragment they were looking for, despite the absence of microbial growth. At these temperatures, spontaneous reactions^[16] degrade the DNA molecule.

In conclusion: Under standard climatic conditions, the degradation of DNA in a dried bloodstain is mostly impacted by microbial growth, which occurs at high ambient humidity levels. So if you want to leave materials behind, do it in a damp undergrowth rather than on a vacant lot, even in the middle of summer. That said, keep in mind a DNA analysis may still be successful.

Analysis of samples containing DNA from multiple individuals

The analysis of samples containing the DNA of multiple individuals is a major challenge for police DNA forensics[9].

It is also a field where lots of research occurs and progress is quick. We will give a small update on what we know about the current state of affairs.

In 2005, there was a study that showed that, on the same sample of mixed DNA, the Experts (with a capital E) did not all come to the same conclusions. What a shame[10].

There are three important factors that influence the success of an analysis of mixed DNA[11]:

• How many individuals contributed their DNA to the mixture? (the more people, the more difficult to analyze)
• How much did each individual contribute to the mixture? For example, if one person contributed a lot, and the others just left a very small amount of DNA (see first graph after this), it will be easier to analyze than if everyone contributed about the same amount (second graph). Also, the smaller the amount left by each person, the harder it is to analyze (third graph).
• How degraded is the DNA? (as usual)

This area has advanced a lot, with new standards and statistical tools to address it. Their tools aren't amazing, but be careful anyway. It all depends on your situation: if you want to anonymize your spoon in custody, you can have it licked by all your fellow inmates, but if you're outside of State captivity, there are more suitable means that we will develop later on (see "The spit, the lighter, the rear-view mirror and the incendiary device").

There are several steps to analyze a DNA mixture:

1. First, identify that it is a mixture.
2. Find the peaks of alleles (like on the graphs).
3. Deduce the number of potential individuals whose DNA is included.
4. Find the contribution ratio of each individual to the mixture.
5. Find all the possible genome combinations.
6. Compare with DNA profile databases.
7. Pray you didn't screw up (apparently, this is important).

Location of the collected DNA

This part has nothing to do with the quality of the analysis itself, but rather with the conclusions that cops or judges can draw from it. The link between the source of the sample and the location where it was collected is what makes DNA evidence incriminating.

Blood, for example, whether found around or at the site of the action, is considered robust evidence. Unlike a cigarette butt, which, if found with your DNA 50 yards from a bank that was trashed, is less incriminating than your blood on the ground in the same location. So for DNA sources other than blood, there is a significant difference[12] between if they are found at the site or elsewhere.

This means that transportable (or mobile) sources of DNA (e.g., hair blown in the wind, bits of skin left on an abandoned bag in the street) are weaker evidence than others (like blood).

We are not saying that if a judge wants to lock you up and his only evidence is a cup with your DNA on it found 100 meters from the site of the action, he will not do it. It's important to keep in mind, however, that just because the cops collected your DNA doesn't mean you're fucked. What they want most of all is a confession, and DNA may very well be all they need to get it.

The genetic fingerprint kept by the cops

Once a saliva sample is taken by a cop (a cotton swab inside your cheek), it is packaged and sent to a laboratory for analysis. Once the analysis is finished, the extracted DNA profile is presented as a table.

What the cops are recording is a sequence of numbers that they hope is unique to each individual. It is not the piece of cotton swab that was used to take the sample. By refusing to give a DNA sample, you refuse to improve the police's knowledge of our genetic information. Every DNA sample taken by the police is a measurement of tiny parts of the genetic information contained in your DNA and not a complete picture. Even if you've already been swabbed, giving another swab means the cops are storing new information about your DNA. If I was already swabbed in 2016, how do I know if the STRs analyzed today are the same as in 2016? Was the accuracy of the analysis in 2016 as effective as it is today?

When in doubt, it's always best to refuse a sample even if you think your DNA has already been collected in some way^[17].

Costs of a DNA analysis

The costs we saw were between 30 and 80 euros for a swab analysis, and around 350 to 420 euros for the analysis of “unconventional samples”, such as DNA on a T-shirt. Note however: we have mostly seen 80 euros in the quotes (rather than 30 euros) for swabs, and mostly seen 350 euros (rather than 420 euros) for unconventional samples.

This information concerns analyses performed in 2018 and 2019.

The spit, the lighter, the rear-view mirror and the incendiary device

DNA can be left at the site of an action in four ways.

Let's imagine. Bibi goes on the attack. The plan: to torch a Porsche on December 31. Bibi places an incendiary device, touches a rear-view mirror with their dirty gloves, spits to relax, and while fleeing under stress, drops their lighter.

At the site of an action, DNA can be deposited directly by falling from our body (the spit) as well as by touching objects (the gloves on the mirror), or DNA can be transfered to the action site via material that is left there intentionally (the incendiary device) as well as material that is lost (the lighter).

So there are two points: DNA that we directly deposit at the site in the moment, and DNA that we transfer there via the material we bring.

Directly depositing DNA at the site of an action

As we have seen, we can let DNA fall from us in two ways. It can fall directly from the body (spitting), or be deposited through contact with objects at the site (the mirror).

DNA is contained in cells* that fall from our body, such as hair, blood, dead skin. These traces can be transmitted from one object to another. Textiles such as wool or large-mesh fabrics allow dead skin to pass through and are very good at picking up the DNA of the people you rub up against (including yourself)^[18]. They should therefore be avoided. A windbreaker is a better idea.

The mirror: If Bibi uses their everyday gloves to go to a riot or action, they increase the chances that their gloves will be covered with dead skin cells that will be left along their path; on their lighter, on a fence or a window, on every object they touch. To reduce this risk, you can use a freshly washed pair of gloves that have not been lying around in a backpack or in a laundry bin. Or even a new pair of gloves^[19].

The spit: To reduce the amount of DNA that falls from the body, during the action we try to be well covered, from head to toe, with clean clothes (machine washed, for example^[20]). This minimizes the risk of hair or pieces of skin falling onto the ground. Avoid leaving your body fluids on or near the site of the action: pee, saliva, etc.

Clean your material before going into action

DNA and fingerprints can be left on material that is taken into action and left behind (the device) or lost near the location (the lighter). There are ways to clean your material in advance to limit the risk of being identified by DNA or fingerprints.

Fingerprints and acetone

The issue of fingerprints is not the main focus of this zine, but we will talk about it very briefly. The contact of our fingers with an object leaves a layer of fat (which comes directly from our skin) on the object. This layer takes the shape of the patterns on our fingertips, and two different individuals have different patterns on their fingertips. Forensic cops are able to pick up these prints on objects and can thus identify people.

To clean an object of fingerprints that may have been deposited on it, we want to degrease its surface. To do this, you can use a sponge soaked in a little acetone and scrub, scrub, scrub. No need to put too big a dose, it is very volatile. Be careful not to inhale too much or swallow it, nor to spread it on your skin. It is toxic and not very good for your body.

On metal surfaces it is more complicated. Oxidation on metal (for example, rusty iron) will have the shape of fingertip patterns, so you will have to sand the surface that needs to be cleaned with sandpaper.

Bleach versus DNA: Clash of the Titans

Another thing that often comes up is that bleach can destroy DNA.

Destruction of genetic information by bleach

The active molecule* in bleach is sodium hypochlorite (NaClO). According to a 1971 study[13], it reacts with all DNA bases via oxidation reactions (chlorination, in this case). An article from 2004[14] quotes this study and states “Exposure of DNA to increasingly higher concentrations of sodium hypochlorite causes cleavage of the strands, breaking the DNA into smaller and smaller pieces, and eventually to individual bases.” In other words, bleach cuts DNA into small pieces. The more concentrated it is and the longer it works, the smaller the pieces will be. This is exactly what we were talking about in relation to the stability of DNA.

If the pieces of DNA, after the action of the sodium hypochlorite, are smaller than the STRs that the cops look for in their samples, then the identification process will be impossible.

The article[14] focuses on determining the effectiveness of bleach in decontaminating bones from the DNA of the archaeologists who collected them. Its conclusion is that immersing an object for about 10 minutes in bleach with 3% sodium hypochlorite cleanses it of DNA contamination^[22].

Concentration of bleach

There are several ways to get an idea of the concentration of the bleach solution. The most commonly used for commercially available bleach products is the “percentage of active chlorine” (noted as % AC). For a fairly basic and concentrated solution, this “percentage of active chlorine” corresponds approximately to the concentration of sodium hypochlorite, i.e. the concentration of interest[15].

The concentration of sodium hypochlorite in the bleach that you will use therefore plays an important role. In stores, the most concentrated solutions are called “bleach extract”, followed by “bleach berlingots”, then “bleach water” and then “Labarraque water”^[23].

How to dilute bleach?

You may want to soak the items to be decontaminated in a bleach bath. This is the technique used in this article[14]: soak the items to be cleaned in a 3% sodium hypochlorite bleach bath for 10 minutes. To prepare a 3% sodium hypochlorite bleach bath, you can obtain 9.6% sodium hypochlorite bleach extract (at the hardware store, for example) and dilute it. Since I want a less concentrated solution, I just need to add the right amount of water. The volume of water to add is given by the following formula:

where, is the volume of bleach extract, is the concentration of sodium hypochlorite in the bleach extract and is the concentration I want to obtain (3% sodium hypochlorite in our example). In this case it gives :

Conclusion: if I add 4.4 L of tap water with 2 L of bleach extract, I get 6.4 L of bleach at 3% sodium hypochlorite for a good decontaminating bath.

A reminder of the precautions to take with bleach

First of all, bleach discolors, so there is a risk of stains on your clothes. Like any chemical product, the more concentrated the bleach is, the more it needs to be handled with care. It is used mixed with cold water. Using a mop with a little bleach in the water with bare hands already has a weird effect, but you won't end up in the hospital. As for the bleach extract, you have to be careful not to put it on your skin, and even more so to avoid getting it in your eyes. If this happens, rinse with a lot of water. Bleach also releases chlorine (a gas that's toxic for our lungs), and breathing too much of it can cause irritation. If this occurs, go outside and get some fresh air. Bleach reacts chemically with certain household products (those containing ammonia, phosphoric acid, hydrochloric acid). These reactions produce irritating, corrosive gases or liquids, so avoid this.

Burn the DNA, burn everything

If you use an incendiary device that works, any DNA will be destroyed by the fire, on the device itself and wherever the fire spreads^[24]. That said, if the device does not work, traces can be found on it by the cops, hence the importance of leaving no DNA traces on the device.

Let's quickly return to cigarette butts being left in custody or elsewhere. A good blast from the lighter on the part of the cigarette your mouth has touched can make this butt useless to the forensic cops.

In the same way, one could clean an object (made of metal for example) with a small blowtorch that works with camping gas. Passing the blue cone of the flame of the blowtorch over the whole object allows the flame to burn the deposits of cells which contain the DNA.

Preventing the use of DNA by cops by targeting their specific practices

DNA analysis by cops involves chemical reactions: it is thus possible to disrupt them, especially using substances that inhibit the PCR process. For example, denim dyes, or certain substances in stamps[1] interfere with PCR. But, as a general rule, cops purify the DNA before analysis, so this is unreliable.

Another strategy is to interfere with the collection of DNA. In this regard, we have read stories about emptying a powdered fire extinguisher into a stolen car, to make collection more difficult, if not impossible.

A protocol for two

Here we propose a protocol for cleaning off the various traces that could be found by forensics and used to establish links between individuals, material and/or actions. In principle, there is nothing to prevent you from working alone, but we find that a group of two people is more efficient. One person can take care of the decontaminated material and the other of the contaminated material. If there are two of us, we can talk to each other during the procedure to share our doubts, questions, etc.

If there are more than two people, there is a greater risk of messing up and leaving more traces than we remove.

To better visualize the protocol, we can divide it into several successive steps:

1. Find a suitable location.
2. Obtain equipment, get dressed.
3. Prepare a surface, a work area.
4. Clean the material you want to clean.
5. Package the material for storage or transport.
6. Pack up the work area, put away what has been used.

It is possible to leave traces at each step of this protocol, so you need to be vigilant throughout the process.

1. Find a suitable location

Arrange to find a quiet spot where you are sure to not be disturbed by people who are not involved in the action or the cleaning and where you believe you won't be overheard. The more people pass by, the more it increases the risks of contamination (you don't want the people who pass by to leave their eyelashes on your things) and the more it increases your stress (and therefore your risk of error).

You should not be too pressed for time (a cleaning session can last 30 minutes or 2 hours, it would be a shame to be interrupted in the middle of work). It's good for the location to be somewhere you haven't been before (to prevent your DNA from already being present), to be clean (dust is full of dead cells and it can make you sneeze, which is not helpful at all) and tidy.

The work surface should be at a convenient height to avoid uncomfortable positions (bent back, bent neck). Also, having your head positioned above the work surface really increases the risk of contamination.

The space should not be too ventilated to avoid air movements which can dirty the work surface^[26].

Once you have your location, the two of you get together, set out your equipment, and get to work.

2. Obtain equipment, get dressed

Equipment

For this step you will need the following equipment:

• Clothes that cover the whole body. You want long sleeves that are ideally tight at the wrists, as well as at the neck so as to not let your hair/skin fall onto the work surface. Clothes must be clean if possible, and should not let fibers fall (avoid fluffy wool, for example). Gloves can be taped to the wrists for longer sessions^[27]. Otherwise, you can try full body paint suits or windbreakers^[28].
• A clean hairnet or shower cap for a confident look (or cap, or windbreaker hood) that covers the head and prevents hair from getting onto the work surface. Clean, single-use hairnets can be a good approach.
• A mask, balaclava, something that covers the mouth, to avoid leaving saliva on the work surface (spit, breath…). Single-use hospital masks may work well^[29]. Goggles or chemical masks can catch falling hairs^[30], but are uncomfortable^[31]. Washing your face beforehand means there will be less dead skin and hair that risk falling off, but this may be nitpicking.
• Thick dish-washing gloves, that go up well above the wrists, so as not to leave any DNA or fingerprints. Note: acetone attacks thin latex gloves, so avoid them. Also, thin gloves can be pierced more easily.

You have to keep in mind that DNA traces can be transported by simple contact. So if you touch a contaminated object with your gloves, and then touch another object, you can contaminate it, i.e. transmit DNA traces onto it.

Decide on roles

This is the moment when you decide the roles of the two people — we will give them names here so that it is easier to follow. The idea is that one person exclusively touches the clean material (i.e. not contaminated by hair, eyelashes, saliva, blood, sweat, whatever) and that the other person takes care of the rest. These roles can't be switched in the middle of the cleaning process (unless you restart from the beginning), so you choose them and stick to them. So, for example, the person who takes care of the clean material we will call Casey (there is a C for Clean). The other person who takes care of the contaminated material we will call Danny (there is an D for Dirty)^[32].

The two people can get dressed together. If possible, get dressed away from the place where the work surface will be. Vigorously shake the clothes before putting them on (to get rid of potential hairs, or pieces of skin which could remain on them, and so won't be brought with you to where you are going to work). Put on the clothes, put on the hat, the hairnet, the mask or the hood that you have chosen. Put on the gloves last.

How to properly put on your gloves?

The gloves must be in individually sealed bags in order to be considered clean. Before putting on the gloves, you should have already washed your hands well, and dryed them well with clean paper towels, for example (avoid shower towels that have not been washed for 6 months). Having short nails isn't a bad idea, if possible (cleaner and avoids damaging the gloves). To have clean gloves, it is important not to touch the outside of the gloves with your hands. So you will pinch the inside of the left glove with the right hand, and put the left hand into it (if right-handed, otherwise reverse), then pinch the inside of the right glove^[33] with the left gloved hand and put the right hand in. There are videos on how to put on sterile gloves for nurses on the Internet that can help. Clean the gloves with bleach^[34].

It is important to wash your gloves with acetone^[25] and bleach if you have doubts at any point. Even if no mistakes have been made, they can be washed with bleach from time to time. In addition, changing acetone-soaked gloves during a long cleaning session can be helpful (being careful not to contaminate anything during this time).

Once gloved, people should not touch themselves, especially Casey^[35]. For example, be sure not to wipe sweat from your forehead or push back your hair (advantage of wearing a cap and having already tied the hair back if necessary). If Casey has to wipe their forehead or scratch their nose, Danny will do it, and will then change their gloves.

3. Prepare a surface, a work area

The idea is to have a clean work surface to be able to put the cleaned material onto it and which will not recontaminate the material afterwards. For this you will basically need garbage bags^[36]. You can assume that the first bag on the roll is contaminated, and that the following bags are clean. You will use garbage bags (rather large and thick) as sheets to cover the chosen work surface. You can also use an actual tarp. If it comes well-packaged from the store^[37] in a plastic bag, it can be considered uncontaminated. To get clean bags:

1. Use a new roll of garbage bags (e.g. 100 L).
2. Danny removes the packaging.
3. Danny takes the roll by the ends and unrolls the first bag.
4. Once the first bag is unrolled, Casey takes the roll (without touching the first bag), and Danny pulls on it to separate the first bag from the roll.
5. Casey can use the following bags and consider them clean.
6. Danny can use the first bag to make a work garbage can (packaging, used equipment, etc. can be thrown into this).

Once the first clean bag is separated and laid down onto the work surface, Casey can put the rest of the whole roll on it. So, when Casey needs a bag, Casey takes the roll and separates a bag, and can consider the bag uncontaminated. The idea is that Casey only puts uncontaminated objects on their work surface. Whether it is clean objects needed for the action, or tools that will be used by Casey during the cleaning. Casey also unrolls one or more bags to make a separate work surface for Danny.

You can set up a work surface on the floor, even if it is not very comfortable, or you can use a large table or a plank on trestles. Be sure to give yourself enough space to work and don't hesitate to make a work surface with several bags if necessary. Basically, the more comfortable you are, the less likely you are to make mistakes.

4. Clean the material you want to clean

For this phase you will need the following material:

• A bottle of acetone^[25].
• A bottle of bleach^[39].
• Sponges or dish cloths in sealed plastic packaging.
• Sandpaper^[25].
• And then… the objects to be cleaned (bottles, pliers, hammers, alien spaceship, etc.)

By “cleaning objects” we mean: “ridding the objects of DNA and fingerprints that might be on them”. For an action you may need to prepare some material, and you'll want this material to be able to be left behind in the case that something goes wrong. If the material in question is clean (i.e. without fingerprints or DNA) you will be a little more comfortable with the idea of the cops coming across it^[40].

During this phase, you clean the objects needed for the action itself, and also the tools you might need to prepare the device: a funnel, pliers, scissors, etc^[41].

If the device is going to require some assembly, you will do it in this phase. For example, if you want to prepare clean molotovs, it is convenient to do the assembly and the cleaning at the same time.

Danny will rub the metal objects with sandpaper^[42] and then use a sponge soaked in acetone to clean all the objects of fingerprints^[25]. Casey will use another sponge^[43] soaked in bleach to clean the objects of DNA traces^[44]. Note that bleach damages metal objects (especially steel objects, which will rust). It's good to keep this in mind, the life span of a bolt cutter that gets bleached once a week will be short. You have to scrub, and put a lot of cleaner: for example, the longer you leave the bleach on, the more the DNA will be degraded. It can be a good idea to put a lot of bleach on, and for it to be left to dry by itself for 15 minutes so that you don't get bleach everywhere.

Danny opens the package containing the sponges (without touching them) and Casey removes a sponge out of the package (without touching the outside of the package)^[45]. Casey uses this sponge for the bleach. Danny will use another one for the acetone.

Danny can start cleaning the tools and objects with acetone, as well as the bottles of acetone and bleach. Danny is very thorough with the sponge, and makes sure to go all over the object, so that its entire surface has been cleaned with acetone^[25]. Once the object is finished, Danny puts it on their work surface (the “dirty” one). Then Casey takes this object and rubs it with bleach, using a different sponge (being thorough once again). Once the object is clean, Casey puts it back on their work surface (the one for decontaminated objects).To return to the molotov example, once the bottle filled with gas is cleaned, Casey can attach the firecracker (that has been removed from its package and placed on their work surface) with a zip-tie (which had the same treatment). The molotov that results is clean; the contaminated parts (the bottle) have been cleaned and uncontaminated parts (the zip-tie, the firecracker) have been added^[46].

The bottles of acetone and bleach are probably contaminated^[47], so it is Danny who picks up the bottle and pours the liquid onto Casey's sponge. The same goes for the gas jerrycan^[48]. Danny can also clean the bottles with bleach so that Casey can use them afterwards. Or we can put a basin full of bleach on the table. You can then wash your gloves in it and/or soak your sponge in bleach^[49].

5. Package the material for storage or transport

Often, there is some time between cleaning and the action. You want to make sure that the material is not recontaminated between its preparation and its use. As a silly example, if you have cleaned your hammer of fingerprints and DNA traces and you leave it lying around your living room, it is likely that people will hold it with their bare hands, and so will leave their fingerprints or DNA traces on it. If the material is not properly packaged to avoid recontamination, cleaning it was useless.

Packaging cleaned material

To package all this, you will need uncontaminated, airtight and sufficiently resistant packaging (depending on the object to be packaged). For example: freezer bags, garbage bags, tupperware, jam jars… Depending on the size and shape of your objects, you will use different packaging. For example, a 20 L garbage bag will suffice to package a hammer, but not a 24″ bolt cutter. For smaller objects, you can use freezer bags with a zip, which are usually sold in a cardboard box. To use them, Danny opens the box, and empties its contents onto Casey's work surface, without touching the freezer bags. For objects that could pierce a plastic bag, a decontaminated tupperware can be used^[50].

A decontaminated object on Casey's work surface is ready to be packaged. To do this, Casey uses clean packaging and places the object in it. Casey is careful to not have their face above the packaging, so as to avoid dropping hairs, pieces of skin, etc.

With garbage bags, Casey holds the bag at arm's length so that nothing falls in. We prefer simple bags, without built-in handles or other gadgets. To close the bag, you tie a knot with the bag or use its plastic tie. To obtain a garbage bag that is considered uncontaminated, proceed as explained in the section on preparing the work surface.

Now that your item is properly packaged, it will not be recontaminated during storage or transport. The outer packaging will be instead. And you can put multiple layers on top of each other, only the outer packaging will be recontaminated.

In concrete terms, when you put your garbage bag in your backpack, the traces that are present in your backpack will contaminate the outside of the garbage bag. This means that if you dispose of the bag in an emergency, it may contain DNA traces. That's embarrassing. So you could keep the outer bag and dispose of its contents. If you used two bags, it might be more practical. It's obviously not easy to quickly open a garbage bag to dump out its contents.

The general idea is that you want to keep the contaminated objects/tools/packaging with you and you can dispose of the non-contaminated ones as needed.

Passing packaged material to others

Once the material has been correctly packaged, it might need to be carried or given to others.

Let's imagine: Danny and Casey have prepared a clean molotov cocktail and packaged it in an airtight bag. It is Bibi who is going to use it. So Casey or Danny will have to give the package to Bibi. Bibi will end up with a package containing an incriminating object inside and possibly Danny's or Casey's (or others') DNA on the outside. If the cops find Bibi in possession of this package and analyze the DNA, they will obviously link Bibi to the cocktail (but for that no DNA is necessary, it's a flagrant crime), but maybe also Danny and Casey if their DNA is on the outside of the packaging!

How to make sure that the package Bibi receives does not contain Danny and Casey's DNA?

When Danny meets Bibi to pass on the package, Danny opens the first bag and Bibi takes the bag inside without Danny touching it. So the package will only contain Bibi's DNA on the outside. If Bibi gets caught with it, the cops will only find their DNA on the package and not that of other people. This is compartmentalization in practice.

The object must be packaged with at least one layer and as many additional layers as there are people in the transmission chain. If Danny gives it to Bibi who then gives it to SuperTomato there need to be three layers of packaging.

6. Pack up the work area, put away what has been used

After the cleaning session, it is necessary to put everything away. A work surface that hasn't been cleaned up is an additional trace of your action. Cops coming across a table covered with gloves, a bottle of acetone, wire cutters, sponges, a funnel (etc.) will make them quite suspicious. It's better to tidy up calmly before the action than in a panic afterwards if something goes wrong.

Basically, get rid of disposable items. The covering used on the work surface, sponges, hairnets, disposable masks, and gloves all go in the trash in yet another bag. You can choose to keep or get rid of tools that you have used (wire cutters, etc.), and you can decide whether to throw away or keep the acetone and bleach bottles (a bottle of bleach in a bathroom is fine). It can be useful to know what bleach and acetone are used for in a house^[52], so you know where to store them in a way that isn't too sketchy, and can be justified if necessary (without going overboard either). If you're going to use water bottles for an action, it is a good idea to get rid of the ones you won't be using. If the cops find your bottles full of gasoline out in the world, you'll feel better if there is no risk of them finding the same bottles full of water in your home.

The only thing left to do is to know what to do with your big garbage bag with all the stuff in it^[53]. Don't just put it in your kitchen or building's garbage can. A quick trip around town or on the road and you'll be able to find a dumpster a ways away from where you did the cleaning that will be great for getting rid of your waste. Be careful, however, if the case is serious, the cops will search the garbage cans around the place of the action or around the homes of the suspects, so put more than two or three streets between you and the compromising material. Another radical solution: the campfire (be careful, acetone is very flammable, do not burn the bottle). Do this quietly, away from people who get offended when plastic is burned.

Glossary

allele: An allele is a form of a gene. Here, we use allele in a broad sense: the allele of a STR is the number of repetitions of the repeated sequence.

bp (base pair): This is the unit of length of a DNA molecule, which counts the number of base pairs (paired nucleotides, like A-T) that the molecule is comprised of.

cell: The cell is the basic element (structural and functional) of living beings.

chromosome: A chromosome is a long DNA molecule wrapped in a series of proteins that serve to maintain the integrity of its genetic information.

enzyme: Enzymes are proteins that increase the speed of chemical reactions in living beings. They can be compared to molecular tools acting on other molecules (to cut them, bind them, etc.)

gene: A gene is a coding sequence of nucleotides, i.e. it is a unit of information for the cell.

genetic profile: A genetic profile is the result of the DNA analysis of an individual.

genome: All the genetic material contained in a cell, and by extension, all the genetic information of the cell.

mitochondrion: The mitochondrion is a sub-compartment of the cell, which serves as its power plant. There is DNA in the mitochondrion (trace of an ancient symbiosis: the mitochondrion was previously bacteria) that we call mitochondrial DNA.

molecule: A molecule is a group of atoms linked together.

nucleotide: Basic unit of the DNA molecule.

nucleus: The nucleus is the sub-compartment of the cell that envelops the DNA and in which transcription takes place. The DNA in the nucleus is called nuclear DNA.

polymorphism: Polymorphism characterizes the ability to occur in different forms.

Appendix A: A little more on DNA (it never stops)

Here, we'll go a bit further on DNA, but also a bit faster. If needed (and wanted), Wikipedia articles are very well done on these subjects.

Quick overview of nucleotides

A single strand of DNA is formed by a succession of nucleotides* (A, T, C, G for Adenosine, Thymidine, Cytidine, and Guanosine). Each nucleotide (in gray ovals in the following diagrams) is formed of a nitrogenous base^[54] (surrounded by a long dotted line), of a deoxyribose (surrounded by a continuous line) and of a phosphate group (surrounded by a small dotted line).

Books have a reading direction: from top to bottom and from left to right. For DNA it is the same. Each nucleotide is oriented, with a side called 5' (phosphate group side) and a side called 3' (deoxyribose side). The DNA strand, which is a succession of oriented nucleotides, is therefore also oriented. This defines a reading direction.

The DNA molecule* itself is made up of two single strands interacting with each other via the complementarity of bases (A–T, C–G) thanks to hydrogen bonds, thus forming a DNA double helix. In the following diagram, we show the complementarity between the bases A and T.

This double helix is then compacted by proteins^[55] to form a chromosome*. This chromosome can itself be protected in the nucleus* of eukaryotes (of which we are a part, unlike bacteria, for example, which belong to… bacteria).

What is a gene?

We are speaking about genes* in eukaryotes, the situation is different in bacteria. A gene is a theoretical unit of heredity. It corresponds to a sequence of nucleotides which codes for a product, which can be either a protein or RNA (Ribo-Nucleic Acid). It is composed of several parts, as seen in the following diagram:

Uses (or not) of STRs

In the rest of the zine, we talked about the so-called non-coding STRs. But there are many different kinds of STRs, with different “uses” (or not).

For example, there may be STRs in the actual coding parts of genes, which will result in repeats of the same amino acid in the protein produced from the gene. STRs can also appear in the regulatory sequences of genes (the sequences that say “please express this gene a little more” or “not this one”). The roles we are discussing here are not well understood, to say the least.

There are also STRs at the end of chromosomes^[57], because every time DNA is copied (for cell division for example), the chromosomes are not copied to the very end. This is one of the reasons why we age. The STRs at the end therefore serve as buffers that can be shortened at each DNA replication without risking damage to the genes. In short, they prevent our genes from being nibbled at during each cell division.

But here we have talked about the evolutionary uses of STRs, but there are also STRs present that do not contribute anything to the organism that carries them. In fact, at the molecular level, mutations are mostly random and are not selected for their evolutionary value. This is the neutralist theory of evolution that was put forward by Motoo Kimura^[58]. This is a complete departure from the subject of the zine, but it is quite interesting to see that 50 years after this theory (which is a consensus among biologists) and in spite of genetic drift, natural selection continues to be spoken of at every turn to justify supremacist ideologies by a pseudo-scientific argument of authority.

Appendix B: Techniques used on DNA during analysis

Prior to analysis, DNA from a sample is isolated and purified. In forensic methods, DNA probe techniques are used to pre-test the presence of human DNA in a sample before proceeding with the actual analysis.

PCR (Polymerase Chain Reaction)

PCR is a molecular copying technique. It can be called amplification. During PCR, not all the DNA present in a cell* is copied: the copying selects for a small segment of DNA using the primers, which are positioned at both ends of the area to be copied and thus delimit the copying range.

PCR Process

The process begins with a piece of DNA that contains the part to be amplified. It is combined with a large excess of DNA pieces that are a few nucleotides* long (called primers). These primers are complementary to a precise region of the DNA that they want to amplify. DNA replication enzymes* (DNA polymerase) and nucleotides, necessary for the synthesis of a new DNA strand, are also added.

1. At 94°C, the hydrogen bridges between the two DNA strands are broken (the DNA is denatured). In the grey rectangle is the sequence that they want to copy many times (to amplify).

2. The temperature is lowered to between 50°C and 60°C. At this temperature, the two strands could reassemble (the hydrogen bonds can be reformed). However, there is a large excess of primers compared to the two initial DNA strands. Most of the hydrogen bonds that will be reformed will therefore be between the initial DNA strand and the primer, which is the black strand with an arrow on the following diagram. Here, we have only made the diagram of one of the two initial DNA strands: exactly the same thing happens on the other strand!

3. The temperature is increased to 74°C, which is the optimum temperature for the DNA polymerase. This enzyme will then replicate the DNA by attaching itself to the primers to start its synthesis. The black circle represents the DNA polymerase.

This cycle can be repeated for as long as they like. Starting from one DNA molecule (double stranded), they obtain 2 double stranded DNA after one cycle, 4 after 2 cycles, etc. So they have 2ⁿ double stranded DNA molecules after n cycles. The number of cycles is normally 28.

Multiplexed STR analysis

PCR is easy to use and can amplify several DNA fragments simultaneously (just add primer pairs). This is called multiplex PCR.

Variants of PCR used

Several types of PCR are used, each with advantages and disadvantages.

• Low Copy Number (LCN) is a DNA profiling method that uses a larger number of cycles for PCR (34 instead of the traditional 28), which makes it both more sensitive to smaller amounts of DNA, and more vulnerable to contamination, among other disadvantages[16].
• Direct PCR is the process of performing PCR without isolation or prior purification of the DNA sample. There is less risk of losing the little DNA present in the sample and less risk of contamination (because there is no isolation or purification), but the subsequent analysis is more time consuming (because there is more chance of ending up with messy mixtures of DNA)[17].

Principles of DNA sequencing

The first sequencing technique invented was the Sanger technique. It uses the principle of chain terminators. A chain terminator is a nucleotide that can bind to a nucleotide during replication, but to which the next nucleotide cannot bind.

It starts with the molecule to be sequenced, a large batch of nucleotides, and chain terminator A nucleotides (i.e. nothing can attach after them). So there is a very large number of copies of the DNA strand they are interested in, and it will be copied with enzymes. At each position where an A must be integrated, some molecules of this very large number of molecules will receive an A terminator. Their copy stops there. In the end, we get a lot of partial copies, with an A at the end. By finding their length, we can deduce all the positions of the A's in this sequence. We can then do the same thing with C, G and T!

There was a time when sequence polymorphism* was used for forensic DNA analysis, i.e., whether the DNA used had the ATTACG sequence or the ATTACC sequence. Now they use length polymorphism, by analyzing the lengths of repetitive sequences, i.e. they try to know if ACG for example is repeated 10 or 12 times in the sample.

Capillary electrophoresis and multiplex analysis

Today, there is also the capillary electrophoresis technique, which allows for much larger potential differences and therefore faster migrations. It is the same principle as gel electrophoresis, but instead of being done on a gel plate, it is done in a capillary. It is a state-of-the-art tool used by forensic police.

Limitations of multiplex analysis and partial solutions

Having several STRs poses a problem for electrophoresis: each individual has a maximum of 2 different alleles* for each STR, so if they analyze 2 STRs at the same time, they have 4 potential alleles. The challenge is to know to which STR each allele belongs to. This can be solved by taking STRs of different lengths. For example, if, for a gene*, all the alleles are between 100 and 170 bp*, and for another gene, all the alleles are between 200 and 250 bp, they can be analyzed at the same time by electrophoresis without risking confusion.

However, they cannot analyze as many STRs as they want all at once, because this implies having larger and larger STR alleles, and the effectiveness of PCR has size constraints. So there are limits to simultaneous STR analysis.

Another solution is to attach fluorescent primers on the different STRs to differentiate them at the time of electrophoresis analysis.

Appendix C: Game of the Goose

Bibliography

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