The PCR method has proved to be very useful in investigation for biology and

medicine. This method consists in making copies of DNA by using an original primer and DNApolymerases, this makes very easy to find the action of bacteria or viruses in the code.
Archeael DNA-polymerases are commonly used, because they are capable of resisting
numerous gradients of temperature cycles. It has also proved to be very helpful in classifying
the population of microorganisms in a certain environment. Anyway, this method is mostly
incapable of detecting low abundance microorganisms with a significantly divergence in their
gene code.
Another way to study a micro-environment is by analyzing the rRNA gene code from
the microorganisms which live in there. Using this kind of studies in an acid mine drainage in
Richmond Mine (California), its diversity was determined. It was composed by Archaea mainly.
Reconstructions of rRNA code from the AMD were done and a different lineage of Archaea was
found and named AMRAN-1 (Archaeal Richmond Mine Acidophilic Nanoorganism). This
discovery was leaded by the detection of an unreported genome fragment in 16S rRNA. After
doing more reconstructions around those pieces of genetic material, a second microorganism
was discovered, AMRAN-2. This one differs from AMRAN-1 by very little, genetically speaking.
Comparing the features of AMRAN-1,-2 with the database it was found that there were
other bacteria that had highly percent similarity with them. Actually AMRAN-1,-2 are part of
wide branching lineage among Euryarchaeota beings.
Another feature of AMRAN-1,-2 is that, they both have insertion sequences in their
16S rRNA code, which is very rare in Archaea and bacteria.
Using primers that target 16S rRNA gene code, samples from Richmond Mine were
studied, and a third AMRAN-type microorganism was found (AMRAN-3). This one would differ
from the other in the presence of insertions in its 16S rRNA code. It has been proven that
AMRAN-lineage organisms actually exist in very different habitats.
Oligonucleotide are short pieces of genetic code (either RNA or DNA). Oligonucleotide
taken from 16S rRNA (AMRAN-lineage ) were fluorescently labeled and the organisms were
mixed with other bacteria and cells. The optic microscope showed that the AMRAN-like
organisms were significantly smaller than the others. To contrast this result, the sample was
filtered into a new sample were AMRAN-2 were the most abundant, but some other (like
AMRAN-1) were present as well. This sample was frozen in special conditions in order to
visualize it with an electron microscope (which helps to determine the morphology and size of
a cell). The result of the experiment was that compared with other organisms such as
Leptospirillum, the AMRAN-type cells were a lot smaller, confirming the visualization of the
optic microscope.
The actual size visualized through the electron microscope is 0.006 m3. This makes
the AMRAN-like organisms the smallest ones in the world (discovered by human beings).
Science says that around 350 ribosomes would fit inside.
The actual size on an AMRAN organism challenges the human definition of life. A good
example is viruses. Some scientists argue about the attribute of life, and they all agree that

viruses are the actual border. Some of them state that they are alive while others are against
that opinion. Some filoviruses have a higher volume than AMRAN-like organisms. So the
question we would have to answer is: What makes an AMRAN-like organism alive and not a
virus?
Life-forms are considered alive when they are able relate with each other, to
reproduce, and to nourish. This is what creates the debate around viruses, because they do
not do all the three requirements. So a first way to understand the alive-behaviour of an
AMRAN organism would be study their way of relating, reproducing and nourishing. If the
three of them are confirmed, we could state they are alive (using the criteria mentioned
before), independently of their possible size or morphology.