Have you ever taken one of those commercial DNA
tests? Oh, right. The ones where you just spit
in a tube and drop it in the mail. Exactly. And
then a few weeks later, you get this beautiful
digital interface telling you where your ancestors
came from. And it gives you all the specific
genetic markers you carry. Yeah. But if you pause
and really look at that data, a massive question
emerges. Right. When a testing company tells
you that your DNA... has certain markers or specific
deviations, what exactly are they comparing your
DNA against? That is the big question. What is
the standard baseline for a human being? Establishing
that baseline is actually one of the most crucial
and, ironically, one of the most complicated
endeavors in modern genetics. Because you need
something to compare it to. Exactly. Without
a fixed point of reference, analyzing millions
of base pairs of DNA across billions of people
is impossible. You need a universal map. And
every map needs a starting point. Right. But
how the scientific community chose that starting
point and what it actually looks like under the
microscope, well, it reveals a lot about how
science actually progresses. Okay, let's unpack
this. We are doing a deep dive today into the
fascinating, slightly flawed, and constantly
evolving yardstick that scientists use to map
human ancestry. Specifically, we're looking at
human mitochondrial DNA, or mtDNA. And exploring
the Wikipedia article in the Cambridge Reference
Sequence. Let's go back to the origin of this
blueprint, which is a story of incredible ambition,
but also a rather notorious laboratory blunder.
To understand the origin, we have to travel back
to the 1970s. A completely different era for
genetics. Oh, absolutely. It was an era of incredible
foundational work, but it was also incredibly
manual. Sequencing DNA wasn't done by dropping
a sample into some automated machine that spits
out data in 10 minutes. Right, they had to do
it by hand. It involved pouring physical gels,
using radioactive tags, and painstakingly reading
bands off X -ray films. At the University of
Cambridge, a group of researchers, led by Fred
Sanger, took on what was a monumental task at
the time. Sequencing the entire mitochondrial
genome of a single human being. Yes. And it was
just one person. one woman of European descent.
This was one. They worked on this immense puzzle
throughout the entire decade, finally publishing
the complete sequence in 1981. And it was an
astonishing technical achievement. It really
was. And to give some scale to what they were
looking at, this mitochondrial genome contained
some 37 genes. Which is tiny. Right, that makes
up only about... 0 .00006 % of the nuclear human
genome. It's a tiny, tiny fraction of the total
genetic material that makes up a human body.
It is a tiny fraction, but mitochondrial DNA
is uniquely useful for genealogy. Because of
how it's inherited, right? Exactly. Because it's
passed down strictly from mother to child, it
doesn't undergo the same complex shuffling that
nuclear DNA does during reproduction. So it acts
as a relatively stable chronological record.
Precisely. When Sanger's team published their
work in 1981, they determined this complete mitochondrial
sequence to be exactly 16 ,569 base pairs long.
16 ,569. Yes. They essentially laid down the
very first track of the genetic railroad, giving
scientists everywhere a standard template. This
became known as the Cambridge Reference Sequence,
or CRS. And having that universal template. allowed
the entire field to accelerate. It gave everyone
a common language. But as technology improved,
other researchers inevitably started to repeat
the sequencing to verify the work or build upon
it. And that is when they noticed some striking
discrepancies. Between what was published in
1981 and what they were actually seeing in their
own labs. Right. The original published sequence
was actually flawed. As other scientists dug
into the data over the next decade, they uncovered
11 specific errors in that initial 1981 publication.
11 errors? Yes. These included incorrect assignments
of single base pairs, which is somewhat expected
given the manual reading of those x -ray gels
we talked about. It's easy to misread a blurry
band on an x -ray film. But it wasn't just a
matter of misreading a gel. The most shocking
part of the initial mapping effort was the physical
state of the sample itself. The contamination?
Yes. The researchers eventually realized that
the sample wasn't purely human. Which is wild
to think about now. It had been contaminated
with bovine DNA and HeLa cell specimens. Contamination
is the invisible enemy in any laboratory, especially
in the early days of sequencing before modern
clean room protocols were standardized. But cow
DNA. I know, it sounds crazy. But the fact that
bovine DNA and HeLa cells, which are a famously
robust and aggressive line of human cells used
in research globally, managed to sneak into the
first complete mitochondrial... sequence is a
testament to how delicate the process is. It's
incredibly sensitive. But the beauty of the scientific
method is that it is fundamentally a self -correcting
discipline. So when they finally scrubbed the
bovine and helicontamination out of the sample
and corrected the manual reading errors, what
did they actually have left? Did the sequence
change drastically? It took until 1999 for a
formal correction to be published. A group of
researchers led by Andrews et al. cleaned up
the sequence, and published what is known as
the Revised Cambridge Reference Sequence, or
the RCRS. Or Revised Version. Right. They formalized
the exact structure, and in doing so, confirmed
that the true length of this woman's mitochondrial
genome wasn't 16 ,569 base pairs at all. It was
different. It was actually one base pair shorter,
16 ,568. Hold on. If they deleted a base pair
from the official record, doesn't that ruin the
numbering system for every single scientific
paper and database published since 1981? It creates
a massive logistical nightmare. By 1999, the
scientific community had been using the original
Cambridge reference sequence for nearly two decades.
That's thousands of papers. Thousands of papers,
global studies, bioinformatics databases, all
built on that specific coordinate system. So
if you shrink the map by one base pair early
in the sequence... Every single coordinate after...
Rendering years of literature instantly obsolete
and incredibly confusing to cross -reference,
it would be like a city deciding to renumber
every single house on a massive avenue. So how
do they handle that missing base pair without
breaking the architecture of modern genetics?
technical debt a workaround yes there was an
extra base pair originally listed at position
3107 in the flawed 1981 sequence it was recorded
as the nucleotide c okay in the 1999 revision
since they knew it wasn't actually supposed to
be there they didn't just delete it and shift
all the numbers down instead they changed that
c to an n just a placeholder a literal phantom
placeholder the n stands in for an unknown or
unspecified nucleotide purely to maintain the
original 1981 numbering system. That is hilarious.
So the sequence goes from position 3106, skips
over the phantom N at 3107, and continues right
along at 3108. So the scientific community collectively
agreed to live with a permanent numbering anomaly
rather than rewrite 20 years of genetic literature.
Exactly. Today, this revised sequence is safely
deposited in the GenBank database under the accession
number NC012920. NC012920. That is the official,
slightly jury -rigged barcode for the baseline
of human mitochondrial DNA. It is. Which brings
us to how this actually affects anyone who has
ever dabbed a cheek swab for a genealogical test.
When you get your mtDNA results back, the lab
doesn't just print out a list of your 16 ,000
base pairs. No, that would be unreadable. Your
results are usually reported as a list of differences
from this revised Cambridge reference sequence.
What's fascinating here is how that reporting
method creates a deeply ingrained psychological
bias. We can call it the yardstick misconception.
The yardstick misconception. Yes. When someone
sees a genetic report that lists their differences
or mutations compared to the reference sequence,
the natural instinct is to assume that the reference
sequence is the original, pure, or biologically
normal human blueprint. The implication is that
your own DNA is a deviation from that norm. It
makes you feel like you are the one who mutated
away from the standard. Precisely. But a helpful
way to reframe the RCRS is to think of it like
the prime meridian. The prime meridian runs right
through Greenwich, England. Right. But there
is nothing geologically zero about Greenwich.
It's just an arbitrary reference point that mapmakers
agreed upon so everyone could navigate. If you
live at 40 degrees west. You aren't deviating
from a perfect location. You're just located
somewhere else relative to that agreed upon pin
on the map. The Cambridge reference sequence
functions in the exact same way. It is purely
a reference sequence, not a record of the earliest
human empty DNA. It is not the biological center
of humanity. Far from it. Geneticists know exactly
where it sits on the human family tree. The reference
sequence belongs to a very specific modern lineage
known as macro -European haplogroup H2A2A1. Wait,
so when a testing company tells me I have a mutation,
I haven't necessarily deviated from the human
standard? I'm just different from this one specific
European woman from the 1970s? That is the core
of the misconception. A difference between your
DNA and the CRS doesn't automatically mean you
carry a rare mutation. The mutation might have
actually occurred in the lineage of the Cambridge
sequence itself. Oh, wow. In fact, the baseline
we use for all of humanity contains genetic markers
that are highly unusual when you look at the
broader global population. The baseline itself
is unusual. Yes. Researchers have pinpointed
seven distinct locations on this genetic map
where our supposed standard baseline includes
rare polymorphisms. Seven distinct spots where
the baseline is actually the global outlier.
Yes. Do we know which ones? We do. These are
specific positions like 263A, the cluster from
311C to 315C, 750A, 1438A, 4769A, 03860A, and
15326A. So a polymorphism is essentially a genetic
variation. Correct. The fact that the universal
yardstick contains seven rare variations means
that for vast swaths of the global population,
comparing their DNA to the CRS involves documenting
differences that are actually just their own
DNA being entirely normal. Exactly. While the
Cambridge sequence is the odd one out. That realization
seems like it would force researchers to rethink
how we compare DNA globally. If you are studying
populations in Africa or Asia, comparing their
DNA to a European haplogroup, H2A2A1 sequence
from Cambridge isn't just arbitrary, it could
be analytically inefficient. It is. If the Cambridge
sequence is just one pin on the map, why not
make others? Well, the scientific community did
attempt to diversify the baseline. Because the
CRS is based on that single European individual,
researchers began using alternative reference
sequences. What were some of the alternatives?
One notable example is the Yoruba reference sequence,
representing the mitochondrial genome of an African
individual. And because it's a different person
from an entirely different lineage, the map looks
different. Yes. The Yoruba sequence has a length
of 16 ,571 base pairs, which is three base pairs
longer than the revised Cambridge sequence. And
scientists didn't stop there. They've also utilized
an African Uganda sequence, a Swedish sequence,
and a Japese sequence as alternative baselines.
The intention behind localizing the reference
points was good, but it created a massive fragmentation
problem. Too many maps. Exactly. Having multiple
different yardsticks, each with its own unique
length and completely different numbering system,
fractures the research landscape. You lose the
universal language that the original Cambridge
sequence provided. Even with its flaws and its
phantom placeholder at 3107, the CRS allowed
a researcher in Tokyo to perfectly understand
a paper published by a researcher in London.
Here's where it gets really interesting. In 2012,
a group of scientists, led by Behar et al., published
a paper proposing a massive philosophical shift
in how genetics handles this mapping problem.
They called it a Copernican reassessment of the
human mitochondrial DNA tree. They proposed throwing
out the revised Cambridge reference sequence
as the standard and replacing it with something
called the RSRS. That Copernican analogy is perfect.
Before Copernicus, astronomers believed the Earth
was the center of the universe, and all the math
used to track the planets was incredibly convoluted
because it was based on an arbitrary starting
point. Right, trying to make the math fit the
wrong center. Behar, et al., argued that using
a modern human, whether from Cambridge, the Yoruba
population, or Japan, as the center of the genetic
universe was equally flawed. The RSRS, which
stands for the Reconstructed Sapiens Reference
Sequence, completely changes the paradigm. So
instead of picking another modern person, what
does the RSRS actually use as its baseline? It
relies on a theoretical model. The RSRS represents
the hypothetical ancestral genome of mitochondrial
Eve. Yes, the theoretical root from which all
currently known human mitochondria descend. That
is a fundamental shift in perspective. Instead
of picking a leaf on the outside of the human
family tree and comparing all the other leaves
to it, they mathematically work their way backward.
Down the branches. All the way to the root of
the tree. They reconstructed what the DNA of
our most recent common matrilineal ancestor would
have looked like. From an evolutionary and analytical
standpoint, it is a vastly more elegant approach.
If we connect this to the bigger picture. Using
the ancestral root as the baseline is incredibly
useful for comparing the changes across different
haplogroups. Because you have a true starting
point. Yes. When you measure differences from
the RSRS, you are measuring the actual evolutionary
path of mutations as they occurred chronologically
over human history. You are tracking the journey
from the root to the leaf, rather than just calculating
the lateral distance between two modern leaves.
It makes tracing the lineage much clearer. It
makes total sense conceptually. But jumping back
to the logistical problem we talked about earlier,
if you change the baseline entirely to a reconstructed
ancestor, doesn't that break all the bioinformatics
databases again? That was the brilliance of the
Behar et al. proposal. The RSRS actually keeps
the exact same numbering system as the old Cambridge
sequence. Oh, really? Yes. They deliberately
maintain the legacy framework to ensure stability
in the global databases, even while fundamentally
changing what the sequence represents at its
core. A structural workaround to save the databases.
Exactly. But a paradigm shift like moving from
a tangible European woman to a theoretical mitochondrial
Eve probably wasn't embraced overnight. It absolutely
sparked an ongoing debate. In 2014, a group led
by Bandelt et al. published a paper pushing back
against the RSRS, arguing strongly to keep using
the revised Cambridge sequence. What was their
reasoning? They made the case for standardizing
notation around the physical known entity. It
seems like a debate between practicality and
theoretical purity. The RCRS is a tangible, physical
sequence. We know it works as a comparative tool,
despite the cow DNA history and the phantom N.
Right. The RSRS is intellectually satisfying
and evolutionarily accurate, but it remains a
reconstructed theoretical sequence. It is a model,
ultimately. So where does that leave the testing
companies today? Do they use the quirky European
yardstick or the reconstructed ghost of mitochondrial
Eve? Commercial companies like Family Tree DNA
have taken a very pragmatic approach. What do
they do? They decided not to choose. They now
report mtDNA results for both the RCRS and the
RSRS. That's smart. They cover all the bases,
providing your genetic differences compared to
both the historical modern standard and the ancient
evolutionary route. It allows users to view their
genetics through multiple lenses. So what does
this all mean? We started this deep dive looking
at a reference sequence, but we found a wild
timeline of human endeavor. It really is wild.
The foundation of modern genetic genealogy started
in the 1970s with a monumental effort to sequence
one single woman's DNA. That sample survived
contamination with bovine and HeLa cells. It
had to be painstakingly revised. It required
a phantom placeholder just to keep the world's
databases from breaking. And it turned out to
contain rare mutations of its own. And despite
all that, it built the modern genetic landscape.
It evolved into a standardized map. And now it
is shifting toward a theoretical reconstruction
of the mother of all modern human lineages. It
is a remarkable narrative. I think the most important
takeaway for you listening to this is about the
nature of scientific truth. How so? Every time
you read a medical claim or look at a genetic
test result or hear about a biological standard,
it is essential to remember that the baseline
of normal is a human construct. It's an agreed
-upon point. Yes. It is a tool we build to help
us understand a chaotic universe. And like all
tools, it is subject to revision, to the realities
of laboratory limitations, and to rigorous, ongoing
debate. The yardstick we use to measure humanity
is always evolving alongside our understanding
of ourselves. It completely changes how you view
a simple pie chart or a list of genetic markers.
You aren't just looking at your history. You're
looking at your history filtered through the
lens of a highly specific, passionately debated,
and ever -changing scientific map. Which leaves
us with a fascinating question to consider as
we wrap up our exploration of this baseline.
Let's hear it. If our ultimate genetic reference
point is actively shifting from a tangible physical
sample like that original Cambridge sequence
to a reconstructed model of the past like the
RSRS of mitochondrial Eve, how might our entire
understanding of human history and our evolutionary
relationships shift if scientists 50 years from
now discover that our theoretical reconstruction
of Eve was slightly off? That is a haunting thought.
When the foundation of your map is a theoretical
ghost, what happens when the ghost changes shape?