Chapter 4: Sources of Biological Evidence

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Welcome back to The Deep Dive.

Today, we're taking a closer look at the microscopic world of forensic biology, specifically that first step, finding and collecting biological evidence.

That's right.

Our mission today is really to understand the huge variety of materials investigators might find.

We're talking fluids, tissues,

all sorts of things.

And we're digging into how and why certain biological bits are so valuable for DNA profiling using insights from forensic science literature like forensic biology.

And that DNA profile, it's just crucial, isn't it?

It can link someone directly to a crime scene.

Or just as importantly, clear and innocent person, absolutely critical.

People probably think of, you know, obvious things like blood stains.

Sure.

But identity gets left behind in ways you might not expect.

Some common items,

things you touch, they can be gold mines.

Okay, let's unpack that a bit.

Like, what are some surprising examples, maybe from that table 4 .1 in the tech?

Right.

Think about a baseball bat handle,

skin cells, sweat.

That could be enough.

Or a licked stamp, an envelope seal.

Exactly.

Saliva is a really common source.

Even something like the residue on a bullet that passed through tissue.

Yeah.

It might pick up tiny fragments of blood or cells.

Wow.

So it's potentially everywhere.

It is.

But, and this is key, the value of that evidence varies wildly.

How much DNA is there?

Is it degraded?

That's where the science gets really detailed.

You need to know what you're looking for and also how likely you are to get a result from it.

Which sounds like a good transition to the, let's call them the standard sources, bodily fluids.

Yep.

The big three for DNA are usually blood, seminal fluid, and saliva.

Okay, let's start with blood.

What's the absolute key thing we need to remember if we're after nuclear DNA from a blood sample?

The key is knowing which cells actually hold the DNA.

See, blood has plasma, the liquid part, and then cells.

Red blood cells, white blood cells, and platelets.

Okay.

But mature red blood cells, the erythrocytes, and platelets.

They actually inject their nucleus as they mature.

They're nullploid.

No nucleus, so no nuclear DNA for standard profiling.

Ah, so it's all about the white blood cells, the leukocytes.

Precisely.

They're the ones with nucleus, packed with DNA.

That's what forensic scientists are isolating, even though white blood cells are, you know, way less common than red blood cells in a sample.

So you're hunting for the rare stuff within the common stuff.

And I guess we should mention specific types, like menstrual blood in sexual assault cases.

Definitely analyzed, yes.

And beyond blood, other fluids are critical, especially in sexual assault cases, what the literature often calls transcellular fluids.

Like semen and saliva.

Semen, saliva, vaginal secretions, absolutely key.

But investigators also consider urine,

fecal matter, sweat.

Even things like earwax,

seramin, or vomitus can potentially yield DNA.

That sounds like a huge list.

There must be some priority, right?

I mean, where does the evidence tell investigators to focus first?

Oh, absolutely.

And this is where you see a really stark difference in reliability.

If you look at the data, say from figure 4 .2 in the text, it's night and day.

How different are we talking?

Massive difference.

Blood samples, you basically get a usable profile almost every time.

Success rate is near 100 percent.

Semen samples, also excellent, around 86 percent success.

Okay, we got it.

But you look at something like transferred evidence,

touch DNA, the stuff we'll get to, the success rate just plummets.

Down to maybe 12 percent.

And hair, only about 18 percent for nuclear DNA, typically.

Okay, so the data screams, get the blood, get the semen first.

Exactly.

That's where you're most likely to get a strong profile.

But what about the DNA that isn't neatly inside a cell?

You mentioned this sort of floating identity.

What's going on there?

Right, that's what we call extracellular nucleic acids.

DNA or RNA that's just outside the cells, floating around in body fluids.

Okay.

Is there a difference depending on the fluid?

Yeah, terminology -wise.

If it's floating in blood plasma, it's often called circulating nucleic acids.

If it's in other fluids like saliva or urine, we tend to call it cell -free nucleic acids.

It might be dissolved or maybe stuck to proteins.

So where does this extracellular DNA actually come from?

Is it just cellular breakdown, waste products?

Some of it might be, but a lot is actually packaged.

Think of them like tiny biological mailbags called extracellular vesicles or EVs.

Mailbags.

Okay, tell me more.

Well, there are different kinds.

You have exosomes.

These form inside the cell within larger compartments, and they get released when those compartments merge with the cell surface.

Then you have microvesicles, sometimes called exosomes.

These basically just bleb off directly from the cell's outer membrane, carrying bits of the cell's internal contents,

including nucleic acids.

You also mentioned a source linked to cell death,

apoptotic bodies.

Sounds a bit grim.

It released to apoptosis.

That's the cell's programmed self -destruct sequence.

When a cell undergoes apoptosis, it breaks down into these membrane -bound fragments called apoptotic bodies.

And these contain DNA.

They do, but the nuclear DNA inside is usually heavily fragmented as part of that self -destruct process.

Not always great for standard profiling.

But what's really interesting is the messenger RNA, the mRNA,

inside these bodies.

It seems to be protected somehow.

Even though the DNA is getting chopped up, the mRNA can remain relatively intact within the epoptotic body.

So the DNA might be toast, but the RNA could still be useful.

Exactly.

That protected RNA might still be identifiable.

And that's potentially huge, because it could tell you what type of cell or fluid the sample came from, even if the DNA itself is too degraded for a full profile.

Okay, so it feels like analysts are juggling two things.

The intact cells with good DNA, and then these extracellular bits and pieces, like vesicles, that might hold different clues.

That's a good way to put it.

Now just to ground us for a second, let's quickly recap the basics of the cells that do contain most of that core profiling DNA.

Mostly we're dealing with two main types relevant here.

You have your standard body cells, your somatic cells.

These are diploid.

They have the full set of 46 chromosomes, two copies of each non -sex chromosome, plus XX or XY.

Then you have the gamete sperm and ova.

These are haploid.

They only have half the set, 23 chromosomes.

And all the DNA isn't just loose, right?

It's packaged up.

Oh yeah, tightly packaged.

The DNA wraps around proteins called histones, forming structures called nucleosomes, which then coil up further into chromatin.

It's incredibly condensed to fit inside the nucleus.

Some parts, eukromatin, uncoil for gene activity.

Other parts, heterochromatin, stay tightly packed.

Got it.

And before we fully leave the cell's interior, we have to mention the mitochondria.

The cell's power plants, they have their own DNA too, right?

Absolutely.

Mitochondria generate the cell's energy, ATP, and they possess their own small circular genome mitochondrial DNA or empty DNA.

And why is empty DNA important forensically?

Two main reasons.

One, there are hundreds or thousands of mitochondria per cell, meaning many copies of empty DNA.

So even if nuclear DNA is degraded or scarce, like in old bones or shed hairs, you might still get empty DNA.

Okay.

More copies means better chance of survival.

Right.

The downside is that it's inherited almost exclusively from the mother.

So everyone in maternal line will share the same empty DNA sequence.

It's less discriminating than nuclear DNA for telling individuals apart, but still very useful, especially for exclusion or linking to maternal relatives.

A reliable backup or sometimes the only option.

Okay.

Let's shift focus now to RNA.

You mentioned mRNA being protected in epoptotic bodies.

It seems like RNA is becoming a bigger player in forensics.

Definitely.

Especially messenger RNA or mRNA.

Remember, the instructions in our nuclear DNA get transcribed into mRNA, which then travels out to the cytoplasm to direct protein building.

The key forensic value here is tissue specificity.

Certain genes are only turned on or expressed in specific cell types.

For example, there are genes active only in salivary glands or only in blood cells or only in vaginal epithelial cells.

So if you detect the mRNA for a saliva specific gene in a stain, then go, you know, that stain contains saliva.

It's incredibly powerful for identify the biological source of the evidence, telling blood from saliva, from semen and so on.

That's really useful.

Now there's another type of RNA getting attention,

microRNAs or mRNAs.

These sound small.

They are tiny, usually just 21 to 23 nucleotides long, but they have a huge role.

They act as negative regulators of gene expression.

They basically help control which genes are active and how active they are.

So they're like dimmers or off switches for genes.

Why is that interesting for forensics?

Well, their patterns of expression, which mRNAs are present and at what levels, can also be very specific to tissue types and potentially even reflect physiological states.

Plus they seem to be quite stable.

So they represent a newer frontier for identifying body fluids, potentially even degraded samples.

How are these tiny regulators actually made?

Is it complicated?

It's a neat little biological pathway, actually.

It starts in the nucleus where a gene is transcribed into a longer primary transcript, prime mRNA.

Okay.

This gets processed, sort of trimmed down by an enzyme in the nucleus called Drosha, creating a precursor called pre -mRNA.

Step one, trim in the nucleus.

Right.

Then this pre -mRNA is shipped out of the nucleus into the cytoplasm by a transporter protein.

Step two.

Yep.

And once it's in the cytoplasm, another enzyme called dicer makes the final cut, creating the short double -stranded mature mRNA.

Step three, final cut in the cytoplasm.

Exactly.

Then one strand of this tiny mRNA molecule gets loaded into a protein complex called the RNA Induced Silencing Complex,

or RISC.

And it's this RISC complex that goes on to target specific mRNAs and block their function.

Fascinating.

So understanding that whole production line helps scientists know how to find and analyze them.

Precisely.

Knowing how they're made, processed, and how they function helps in developing methods to detect them reliably in forensic samples.

Okay.

We've covered fluids, extracellular bits, RNA.

Let's move to the more solid stuff.

Tissues that often turn up at crime scenes.

Skin, hair, bone, teeth.

Let's start with skin, the source of the famous touch DNA.

Right.

Skin has layers, as you know, epidermis, dermis, subcutaneous.

The evidence we're usually interested in for touch DNA comes from the very top layer of the epidermis, the stratum corneum, or cornified layer.

And what's that made of?

It's mostly made of cells called corneocytes.

These are basically dead skin cells.

They start deeper down in the basal layer as living keratinocytes.

But as they move up, they differentiate, lose their nucleus and organelles, and become these flattened dead cells packed with periton.

And they're constantly being shed.

Okay, hold on.

If they're dead and they've lost their nucleus, how on earth do we get nuclear DNA profiles from touch evidence?

That seems like the central challenge.

It absolutely is the challenge.

And the answer is it's not usually from intact nuclei within those corneocytes.

The DNA comes from few potential sources, often in combination.

Such as?

One,

remnants.

Small fragments of nuclear DNA might still persist from when the nucleus partially degraded during that differentiation process.

Not ideal, but sometimes enough.

Okay, fragments.

What else?

Two, cell -free DNA.

Remember that?

DNA floating around.

Sweat glands and their ducts open onto the skin surface.

Cell -free DNA present in sweat can be deposited along with these shed corneocytes.

Ah, so DNA from the sweat itself.

Potentially, yes.

And third,

you might occasionally transfer a few actual nucleated cells.

Maybe from deeper layers or from the sweat glands themselves.

Especially with more friction or pressure.

But mostly it's those first two.

It's often very low quantity, fragmented DNA,

highly variable.

Which explains why collecting it effectively is so important.

You often hear about the double swab technique.

Yes, that's standard practice for touch DNA.

You typically use one swab, often cotton, lightly moistened with sterile water or buffer to collect the sample from the surface.

Then you immediately follow up with a second, dry swab over the same area.

Why two?

The idea is a moist swab helps lift and transfer the cellular material and DNA.

And the dry swab helps collect any remaining material and absorbs excess moisture, potentially concentrating the sample.

Maximizes recovery.

Makes sense for such trace amounts.

Okay, moving on to hair.

What's the key for getting DNA from Well, hair structure has the medulla, cortex, and cuticle.

For nuclear DNA, the best source is the hair root, specifically if the hair was actively growing when it was removed.

That's the antigen phase.

Exactly.

In the antigen phase, the cells in the hair follicle, the matrix cells, are dividing rapidly.

So a plucked antigen hair often comes with follicular tissue attached to the root, which is rich in nucleated cells and thus nuclear DNA.

But crime scene hairs are often shed naturally, right?

Not plucked.

Very often, yes.

Naturally shed hairs are typically in the telogen phase, the resting phase.

These are called club hairs.

They have a hard keratinized root, and they generally contain very little, if any, nuclear DNA.

They've detached from the follicles dividing cells.

So a shed telogen hair is often bad news for nuclear DNA profiling.

Often, yes.

Which is why mitochondrial DNA, empty DNA, becomes so incredibly important for hair evidence.

Because it survives better.

Much better.

Remember those high copy numbers.

Plus, the empty DNA molecules are physically protected within the keratin protein matrix of the hair shaft itself.

That tough structure shields the empty DNA from environmental damage and degradation far better than it protects nuclear DNA in a shed root.

So empty DNA is the go -to for telogen hairs?

It's often the only viable option.

But there's a complication we need to mention with hair empty DNA.

Heteroplasmy.

Right.

What exactly is that?

Heteroplasmy is when an individual has more than one empty DNA sequence, either within a single hair, or between different hairs, or even within different tissues.

How does that happen in hair?

The thinking is it might relate to the pigment cells, the melanocytes in the hair follicle.

These cells actually transfer pigment granules,

and potentially some of their mitochondria along with them, to the developing hair cells, the keratinocytes.

If the melanocyte empty DNA sequence is slightly different from the person's primary empty DNA type, you can end up with a mixture in the hair shaft.

So it's something analysts have to be aware of when comparing empty DNA from a crime scene hair to a reference sample.

Absolutely.

You might see minor sequence differences that are due to heteroplasmy, not because the hairs are from different people.

It requires careful interpretation.

Okay, let's tackle the really tough stuff.

Bone and teeth.

Usually used when dealing with decomposed remains or mass disasters, right?

Exactly.

They are often the last biological tissues remaining that can provide identifying DNA.

Why is bone so resilient?

It's essentially a mineralized matrix.

It's hard, dense.

The DNA itself resides primarily within specialized bone cells called osteocytes, which are embedded within tiny spaces, lacunae, in that calcified matrix.

How many are we talking?

Quite a lot, actually.

Around 20 ,000 osteocytes per cubic millimeter of compact bone.

So the potential for DNA is there.

But degradation is still a huge issue.

Oh, yes.

Environmental factors, temperature, humidity, soil chemistry,

microbes, they all take a toll.

The DNA inside those osteocytes will degrade over time.

The challenge is getting enough quality DNA out.

And getting it out isn't simple, I imagine.

You can't just swab a bone.

Definitely not.

That hard mineralized matrix is a barrier.

You have to physically break down the bone structure and then chemically break down the matrix to release the DNA from the osteocytes.

How is that done?

It usually involves physically grinding the bone into a powder first to increase surface area.

Then a critical step is decalsification.

You use chemicals, often EDTA, to bind and remove the calcium ions, essentially softening or dissolving the mineral part of the matrix.

Okay.

Get rid of the calcium shield.

Right.

And then you follow that with enzymatic digestion, typically using proteinase K, to break down the remaining proteins, including those holding the cells together, to finally liberate the DNA.

It's a pretty harsh process.

It sounds intensive.

Now, what about teeth, often considered even better than bone?

Generally, yes.

Teeth are incredibly durable.

The bulk of the tooth is dentin, which is mineralized.

And it's covered by enamel on the crown, the hardest substance in the human body, and cementum on the root.

This structure provides amazing protection for the DNA inside.

Protection from what?

Environmental damage, heat, microbial attack,

much better than bone, often.

And where is the DNA located within a tooth?

Well, there's the dental pulp in the center, which is soft tissue rich in cells and DNA, but that degrades relatively quickly after death.

So if the pulp is gone?

You still have DNA within the hard tissues themselves.

In the cementoblasts, the cells that form the cementum layer on the root surface, and also within tiny channels running through the dentin, which contain processes extending from cells called odontoblasts.

These processes contain mitochondria and maybe some nuclear DNA remnants.

So DNA is locked inside the hard part, too?

Locked inside, yes.

And getting it out requires similar steps to bone.

You usually have to cut the tooth into sections using specialized saws or rotary tools, then clean it thoroughly, and then, again, that crucial decalcification step to dissolve the mineral matrix, followed by protein digestion to release the DNA.

Wow.

So both bone and teeth rely on breaking down that mineral fortress.

That's the key hurdle for extraction, yes.

This has been quite the journey.

We've gone from reliable fluids like blood, looked at the tricky nature of touch DNA, explored the emerging world of RNA markers, and ended up with the sheer resilience of bone and teeth, a really broad spectrum of forensic identity sources.

It really is.

And if you step back and think about the bigger picture,

the level of detail we need to understand is just incredible.

It's not just about finding any DNA, it's about knowing the specific cell type, understanding how DNA degrades or gets packaged in something like an apoptotic body, knowing the exact enzyme pathway that creates a microRNA, or figuring out the chemistry needed, like decalcification, to get DNA out of a tooth.

It goes way beyond just matching patterns.

Absolutely.

True critical thinking in forensic biology means appreciating the how and why behind that tiny biological trace surviving and being identifiable.

It's about understanding the journey of those molecules.

That's the real deep dive.

Astounding complexity, astounding resilience.

Thank you so much for walking us through this intricate landscape of forensic sources of identity today.