Episode #001 – Transcript

Cast:

David Bakker as Dr. Steven Strangelove

Caleb Wale as host Reginald Woods

Ella Alfonso as Eleanor Lestrade

Outline:

  • Set up podcast to investigate the various methods of DNA analysis in crime
  • Spin the story of a crime that occurred in Kelowna, BC in the year 2018
  • Discuss PCR, RFLP, VNTR and STR analysis and include comparisons
  • Explanation of gel electrophoresis
  • Discuss the CODIS system & 13 loci + Amelogenin

Script:

HOST:

Good morning listeners, and welcome to 95% Certain, a podcast about recent science developments and its effects on human society.  I’m Reginald Woods.

 

This week on the show we are taking a deep dive into DNA forensics.  We will be examining their reliability, privacy and ethical concerns, while also looking at the human effects on real people affected by this emerging criminal science. A variety of methods are currently employed by the judicial systems in North America for different samples of evidence available at the crime scene at hand. In this episode we will be examining a crime that was committed in a campus laboratory at the University of British Columbia in 2018.

 

A number of details were known about this grisly crime, but DNA analysis proved to be pivotal in the solving of this particular crime. On the night of March 2nd, 2018, the body of a graduate student named Larry Wilson was discovered in a third floor laboratory where he had been conducting research for his master’s thesis.

The body was found on the floor with a graduated cylinder lodged down the throat. This piece of glassware was analyzed and contained trace amounts of highly concentrated Hydrogen Chloride, a volatile acid. The cylinder also had skin cells from the assailant present that were collected for DNA analysis.

It appeared that Larry had attempted to defend himself, as a large glass pipette was also found lying close to his hand broken in half with blood coating the jagged edges. This pipette had been used as a stabbing weapon, as Larry tried to free himself from his attacker.

As a result, there were large droplets of blood on the floor trailing out of the room that were also collected for lab forensics.

This murder was committed between 8:39 pm when five of Larry’s colleagues left him behind in the lab to finish his work, and 9:03 pm when the body was discovered. During this time, 4 individuals were seen leaving the 3rd floor of the building where Larry’s laboratory was located.

1)The first suspect was Larry’s girlfriend Janessa Wright, who claimed to have been stopping in to check on her boyfriend before leaving campus for the night.

2)The second suspect was Larry’s supervisor, Ludwig Bennison. Ludwig had allegedly been looking for a report from Larry that had been requested a week earlier.

3)The third suspect was one of Larry’s colleagues, Brian Mulroney, who had supposedly forgotten his laptop in lab and returned to grab it.

4)The fourth and final suspect was Fiona Jensen, an undergraduate student who claimed to have been looking for help on an assignment from her class in which Larry was a TA.

Each suspect was interrogated and had blood and skin cell samples taken to compare against the evidence left at the crime scene. DNA analysis for this case was conducted by Dr. James Dolman who used three different methods of analysis to determine the culprit for Larry Wilson’s murder. Dr. Dolman cannot speak with us due to his subjective involvement with the case as it has not been resolved in court at this time.

 

HOST:

Featured in this week’s program is an interview with Dr. Steven Strangelove, a BioChemistry Professor Emeritus at the University of Mendoza.

A professor of over 30 years, Dr. Strangelove has watched the development of DNA forensics throughout its history, as well as significantly contributing to its ongoing development.

In addition to his research work, he has taught several of today’s leading forensics analysis, including the investigator of this case, James Dolman. Strangelove has also been called upon in numerous trials for his expert witness testimony.

Dr. Strangelove, glad you could join us!

DR STRANGELOVE:

Happy to be here to shed some light on this very important subject.

HOST:

Let’s start by going through the origins of DNA analysis.  We tend to have a very scientifically literate audience, so we can breeze over most of the DNA basics and jump right into action. What was the first technique used, and how does it work?

DR STRANGELOVE:

A great question Reginald, and thanks again for having me. The original method for DNA analysis is Restriction Fragment Length Polymorphism.

HOST:

. . .That’s quite a mouthful! . .

DR STRANGELOVE:

Indeed it is, so we just shorten that to RFLP. Keeps everything easier. The basis of this technique is to use molecular scissors, or restriction enzymes, to cut DNA at specific known sequences in the base code.

HOST:

So that would be at a known sequence of As, Gs, Cs, and Ts.

DR STRANGELOVE:

Exactly! This analysis type uses the fact that everyone’s DNA varies just a little bit with the location of these specific sequence, so when the restriction enzymes are added, the remaining DNA has different length chunks depending on the person. This means that based on the length of the cut up chunks of DNA, individuals can be differentiated from each other.

This is inherited from your parents, with your different restriction sites, resulting in different length segments of DNA after the restriction enzymes cut the DNA up. You have the same heterogeneous or homogeneous lengths depending on whether your parents had identical restriction sites or not.

HOST:

Oh fascinating! It’s really as simple as that? All you have to do is add some molecular scissors, and BOOM! You’ve caught the criminal?

DR STRANGELOVE:

Unfortunately, it isn’t quite that easy. While the restriction endonucleases cut the DNA, it doesn’t separate it or allow easy comparison. All the DNA is still pooled up together in a tube, and I could tell just as much from that as the average Joe on the street could; diddly squat.

And don’t forget, not only is there one tube of DNA fragments from the crime scene that needs to be analyzed, you need to compare it with the samples from all the suspects. You need to collect blood samples, containing large quantities of high quality DNA from all the crime’s suspects, and prepare their own separate tubes of the DNA fragment soup. A laboratory technician then has to take all of these tubes of DNA fragments, and use a system called gel electrophoresis to separate them and allow their comparison.

HOST:

Gel electrophoresis . I am trying to break one down right now. “Gel” is fairly obvious, it must be using some sort of molecular matrix, uuhh “electro” means that it involves electricity, but “phoresis” has me a bit stumped.  How does this process work?

DR STRANGELOVE:

You’ve got it exactly right there Reginald. At its most basic, gel electrophoresis is using an electric current to disperse, that’s the phoresis part is disperse, the DNA samples using electricity through a gel matrix.

You know the old saying that opposites attract?

HOST:

Haha yeah my ex definitely used that one on me in my university years, I didn’t find it to hold much truth in that case though.

DR STRANGELOVE:

While it might not be true with love based on your story, but it is the case with both magnets and also electrically charged molecules. Something that is negatively charged will try to move towards a positively charged object, and vice versa. Likewise, if you’re the same as something else, or have the same charge, you’ll be repelled from it. So when it comes to DNA, which is an overall negatively charged molecule because of the chemical species that make it up; it will be attracted towards something that is positively charged.

So since our goal is to separate different chunks of DNA, we need something beyond just the charge to separate them. Because all the DNA has basically the same charge. So instead you need to both add that external charge and basically put the DNA in a maze of ropes, made out of the chemical agarose, so that bigger pieces take longer to find a way through the maze than the smaller pieces do. Then you add the big external positive charge, or voltage difference, to push and pull the DNA through the maze.  If you want to increase the separation, all you have to do is add more agarose to the maze, or vice versa if you want to decrease the separation.

This is because the smaller pieces of DNA are able to navigate through smaller holes in the maze, whereas the larger pieces get stuck more often as they are not able to fit through the smaller holes. This is actually quite a common separation technique in both chemistry and biology laboratories.

HOST:

Yes, I’ve heard of couple gel, type, methods in the lab. I think I am following you. All that you need to do in gel electrophoresis is put DNA in the middle of a maze, then add some electricity to push it through. This is what separates the DNA by its size, correct?

DR STRANGELOVE:

Exactly, you got it!

HOST:

That all makes sense, but I still don’t understand how this allows you to compare the suspect samples with that found at the crime scene. Or how you visualize the spreading because DNA is so small that it is invisible to the naked eye, is it not?

DR STRANGELOVE:

I am so sorry Reginald, you are right. I forgot to mention that prior to adding the DNA to the gel electrophoresis matrix, you first need to bind a loading dye to it. This helps to make sure that the added DNA both sinks into the agarose matrix, as well as allowing the analyst to see it. Under UV light it fluoresces, enabling easy viewing of the DNA bands. A DNA Ladder is also added to the gel matrix which allows an approximate determination of DNA size, say for compiling into a database for further analysis.

HOST:

Sorry for intruding in the midst of your explanation Dr. Strangelove, but I would just like to inform our viewers that the photos of the actual gel electrophoresis in our case study are available on our website at DavidBakker.me/95percent. That’s David Bakker with 2 K’s. If you didn’t catch that the first time, that’s DavidBakker.me/95percent.

Sorry about the interruption there Dr. Strangelove, please continue.

DR STRANGELOVE:

Not at all Reginald, I am glad your listeners can also see this valuable information in addition to this podcast.

Back on point, this visualized DNA is referred to as the DNA fingerprint. If the DNA found at the crime scene has the same bands in the same location as those of a suspect, we know they committed the crime. Likewise, a suspect is exonerated if his DNA fingerprint does not match that found at the crime scene.

HOST:

So RFLP when coupled with gel electrophoresis to visualize it sounds like an almost foolproof system, is there a catch? If there isn’t any catch, why did scientists bother with pursuing any other DNA analysis systems?

DR STRANGELOVE:

Yes, unfortunately there are some significant disadvantages to the RFLP system; the most important of which is that this method requires a large amount of sample DNA to be analyzed. It also has to be really high quality DNA such as directly from a blood sample. While the huge advantage is that it is very simple to do, it is almost never the case in most crimes that the perpetrator leaves behind a large pool of blood to be easily analyzed, so this method is all but obsolete. Thus we as scientists had to pursue better, more sensitive methods. These newer methods can use smaller amounts of DNA, and lower quality DNA that might be from other sample sources as well, that could potentially even be a little bit degraded.

HOST:

I see, what other methods for these lower quality sources of DNA have been developed since use of RFLP has been phased out?

DR STRANGELOVE:

There have been two primary methods developed for lower quality DNA samples, they are called VNTR and STR, at least colloquially. The method developed for analyzing the smallest and lowest quality samples of DNA is STR, or Short Tandem Repeat analysis. This method looks at tiny gene segments that are only 2-9 base pairs long, that’s your A’s, C’s, G’s and T’s again.

HOST:

Ok so judging from the name this is repeated segments of the same 2-9 base pair sequence?

DR STRANGELOVE:

Yes you’re on the right track! These segments are repeated different amounts of times between individuals based on genetic inheritance passed down from their parents, for example if an STR region was located on the 18th pair of chromosomes, one is from the mother and one from the father. Since humans are diploid organisms and thus have duplicated chromosomes. The amount of repeats on each chromosome may be the same or different between the two different 18th chromosomes, such as having 4 repeats on the mom’s duplicate and 7 on the one inherited from the dad.

When we compare the number of repeats between chromosome pairs at a number of different sites, it becomes increasingly unlikely that two individuals, even relatives, can share the same profile of STR regions throughout their entire genome.

HOST:

That sounds like a very reliable method. What is this other method… VN..T…Q?

DR STRANGELOVE:

Very close, you almost got it! The other method is VNTR, or Variable Number Tandem Repeats analysis, which follows the same premise as STR but uses longer gene segments in the repeats that are looked at. Repeats in this method are typically 10 – 100 base pairs long, whereas in Short Tandem Repeats, or STR there are only 2-9.

Both this method VNTR and STR can use a technique called polymerase chain reaction, or PCR for short, to replicate the desired region of DNA that contains repeats.

This allows you to take a very small sample of DNA taken from the crime scene and replicate it up to a point where it can effectively be used for analysis. In both methods the repeat regions are isolated by cutting the DNA at specific sites using Restriction Endonucleases, which we discussed earlier in RFLP. They are basically the molecular scissors that cut the DNA.

HOST:

Ok so which of the two repeats methods is better?

DR STRANGELOVE:

VNTR provides a slightly better differential result than STR, so fewer sites need to be examined to create an individual profile. The drawback of VNTR is that it needs a decent quality sample with at least a couple hundred base pairs to be able to analyze properly, whereas STR can use slightly degraded DNA and very small samples as well. So which one is better depends entirely on what samples are found at the crime scene.

HOST:

Oh very interesting! Could you clarify how this PCR process works? It just replicates the gene of interest over and over indefinitely?

DR STRANGELOVE:

Yes of course, sorry I didn’t explain that very well at all! As long as you keep adding the proper elements, PCR basically just doubles the amount of DNA with each cycle. So the first thing you need for this process is a sample to be amplified that has been cut by the restriction endonucleases we talked about a few times already. This chunk that has been cut out will now act as your template strand.

Next we’ll need to add some of the proper primers, which are very specific to bind the template at the beginning of the gene we are interested in replicating.

These primers also allow the binding of an enzyme called DNA Polymerase to the template. A PCR version of this enzyme called, Taq Polymerase is heat resistant and used for this function. Finally, you need to add all the different bases, your A’s, T’s, G’s and C’s, called dNTPs in this context to be present as building blocks for the new DNA being produced. A bit of MgCl2 is also needed as a cofactor.

It’s basically just like cooking, you add all the stuff and then you heat it up, and then you cool it down. Except we do it over and over again because we want to keep on making more.

HOST:

Okay, so once we have all the ingredients in the pot, how does this heating up and cooling down process occur? What are the details associated with that?

DR STRANGELOVE:

Once you have all the components needed for PCR in the pot as you put it, the reaction can proceed in cycles. Each cycle involves 3 stages. The first stage is to heat the mixture until the template DNA is denatured into 2 single strands, which is followed by the second step where the primers can then anneal to the template as the mixture cools. In the third step, Taq polymerase attaches to the primers and produces a complementary strand for each of the exposed single stranded pieces of template DNA. These steps can then be repeated as many times as needed to amplify the DNA to the desired quantity for forensics testing, or in fact PCR can be used for other purposes in many other fields as well.

HOST:

Sorry for taking it back to the very beginning, but I’m also curious about how DNA is able to be extracted directly from tiny samples such as skin cells. What kind of chemicals or techniques are able to pull DNA out of the cells and expose it before restriction endonucleases are used?

DR STRANGELOVE:

No need to be sorry there is nothing wrong with a little curiosity! A well established method for extracting DNA from small samples such as skin cells, or small stains of dried blood or semen, is using a compound called Chelex 100. This compound is a chelating resin and has been proven to be more effective at extracting these samples than phenol-chloroform compounds that were used in the 1980s and early 1990s.

This was proven in a study by Walsh et al., published in the journal of BioTechniques. Once the DNA has been extracted in this way, fragments are free in solution and ready to be digested by restriction endonucleases!

HOST:

Oh I see! Thank-you for answering that last question, it was great to have you on the show.

Wow, that process sounds revolutionary! Thank you for amusing my curiosity, it’s incredible to hear about the specifics of how these techniques work at the molecular level; to augment DNA and allow us to identify an entire human from one drop of blood.

Thank-you so much for your time Dr. Strangelove.

DR STRANGELOVE:

My pleasure, thank-you very much for having me.

 

-SHORT BREAK-

 

HOST:

To further our understanding of the subject matter, we have invited Ms Eleanor Lestrade to the program. After taking an undergraduate degree in statistics at Oxford, Ms Lestrade then studied law at McGill. She currently works at a prestigious law firm, based in London, Ontario.

Welcome to 95% Certain Ms. Eleanor Lestrade.

ELEANOR:

Thank you for having me. I was listening to your previous guest Dr. Strangelove, and I thought he summed up today’s analytical techniques brilliantly.

HOST:

We’ll get straight down to business.  Over a dozen countries, including Canada and the USA, use a software system called the Combined DNA Index System to incorporate genetic information both collected at crime scenes and from criminals, which is compiled into government databases.

DNA analytics has come under some scrutiny from the public for potential invasions of privacy. Especially since your DNA can be legally collected if you are accused of a violent crime. Your DNA sample can be collected before you are convicted as well.

This is a matter of concern for privacy experts, since after all, your DNA is what makes you, well, … you.  There are some who argue that collecting DNA and storing it in government databases is a rampant invasion of privacy.  What are your thoughts on the matter, both personally and professionally?

ELEANOR:

Yes, it is true that the Combined DNA Index System, or CODIS for short, is used to analyze and compile DNA identifying information in many countries.  However, the privacy concerns are not as severe as they may seem.

Large chunks of your DNA do not code for proteins, and thus are not actually expressed. The CODIS system only analyzes these portions of your DNA, so that no medical information is determined or saved on the criminal suspect. These non-coding DNA sequences are called anonymous sequences.

So while some DNA data is collected in a database, it is not medically interesting DNA such as that collected by commercial DNA analysis companies such as 23andMe.

HOST:

That does provide me with some reassurance for sure.  Not that I have committed a crime, it is reassuring to know that criminals are treated with a certain level of respect.

How does the CODIS system work in relation to the DNA analysis techniques described earlier by Dr. Strangelove?

ELEANOR:

So the VNTR and STR described by Dr. Strangelove are used as he described.  But then we take that information and store it in a database. When Dr. Strangelove described the inheritance of the different restriction sites, he used terms like homozygous, where you have two of the same gene at one locus, or heterozygous where you have two different genes.

The CODIS system tests the DNA sample at 13 different loci, or location in the genetic code. If only one location was tested, lots of people could potentially match each other, which would make identifying one individual useless. However, with each additional location tested, the likelihood of a match between distinct individuals decreases. Statistics shows that with 13 different loci, and only 7 billion people on the planet, the chance of a random match is absolutely miniscule.

HOST:

Okay so the system is designed in such a way that the random match probability is minimized, but there is a still a small chance of false match?

ELEANOR:

Yes, that is correct.  While there is still a small chance of a false match, that probability is lower than you getting struck by lighting tomorrow.  The random match probability, or RMP, is carefully considered and takes into account allelic frequencies, the well established genetics Hardy-Weinberg equilibrium, and the statistical product rule.

Together, this all but ensures that the probability of a random match is all but nothing.

HOST:

Okay so let’s just sum this up. CODIS minimizes privacy invasion by using portions of the DNA that do not code for medical information, and minimizes the chances of a false match by using 13 different sampling sites in the DNA.

ELEANOR:

You have got it exactly right.  There is one other brilliant little addition however. The CODIS system also adds in a 14th test, which is for the amelogenin gene which allows for determination of biological sex.

HOST:

This sounds like it all works great in theory, but is there practical evidence that proves the system actually works? And that we aren’t jailing innocent people with this technology?

ELEANOR:

Yes there are many studies with large sample sizes that have been published recently. For example, Yossi et al., investigates different techniques of the CODIS system to identify mass disaster victims (2018).

These large sample size studies with good matching results show that even with groups of similar genetic descent can properly differentiate individuals from each other. Applying this understanding to criminal law indicates the validity of the CODIS system.

HOST:

Wonderful. Your view on this subject has been invaluable Eleanor, thank-you for your time.

ELEANOR:

My pleasure Reginald, thank-you for having me.

 

Conclusion:

HOST:

We now return to the murder of Larry Wilson in Kelowna, where suspects had been narrowed down to 4 individuals based on the conducted police investigation. At this point in the case, there was little witness or circumstantial evidence to separate the suspects.

These individuals were all required to have a skin and blood sample taken which could be used to conduct DNA analysis by Dr. James Dolman who was mentioned previously. Dr.Dolman provides consistent, reliable results in all his case files. Each suspect was numbered 1-4 in the respective order of Janessa Wright as number 1, Ludwig Bennison as number 2,  Brian Mulroney number 3 and Fiona Jensen number 4.

Dolman used the 3 methods explained to in my interview with Dr. Strangelove: RFLP, VNTR and STR. He used the VNTR technique to eliminate the detectives at the scene from being analyzed, using a cheek swab from each detective as reliable and non-invasive sample.

Using the blood samples from the crime scene and each suspect, he was able to run RFLP analysis by digesting the samples with restriction endonucleases, then running the samples on a gel. He was also able to use the skin samples from suspects and the crime scene to run STR analysis. He amplified these samples with PCR, then he was able to run these on a gel as well.

Images of both gel electrophoresis trials can be found on our website at DavidBakker.me/95percent. The VNTR tests for investigators at the scene were inconclusive, but further circumstantial evidence eliminated them from being suspects of this murder.

In both the RFLP and STR analysis, the evidence concluded that the DNA of suspect 3, Brian Mulroney, matched that of the samples left at the scene of Larry Wilson’s murder. Drawing from this DNA evidence, Brian Mulroney was identified as the primary suspect and is now facing trial for the murder of Larry Wilson.

This murder did not appear planned, and the motive is still largely unknown for a fellow graduate student who seemed quiet and friendly. An impulsive decision proved to be deadly in this case, with a common lab acid finding itself used as a murder weapon.

 

Thank you listeners for joining us on this episode of 95 percent certain. I hope that we were able to provide you with some insight into the world of DNA analysis and forensics, a fascinating field that has vastly changed the way justice is enforced in the modern age. Goodbye for now. I’m 95 percent certain you will join us next time on the show for another dose of tangible science to power your day. I’m Reginald Woods.