Mitochondrial DNA Test Results

Original article posted on November 7, 2009

I’m supposed to be studying right now, but I can’t help myself. This document dump is too addictive. I’ve decided to step through it in sections of similar testing. So the first interesting group of test results we are faced with on this released discovery are mitochondrial DNA results.  In a previous entry, here, we discussed nuclear DNA test results that have been released.  Nuclear DNA refers to the DNA within the nucleus of your cells.  It is what makes “you” “you”.  We discussed genotypes and phenotypes and all that neat stuff, and went over alleles and what they represent.

In mitochondrial DNA testing we have to travel outside the nucleus of your cells, and we start dealing with a second group of DNA that resides in your body.  It is the DNA of the mitochondria.  The region of your cell that lies outside the nuclear wall is referred to as the cytoplasm.  Your cells are made up of organelles which can be viewed as miniature organs within the cell itself.  So like your body, on a macro scale, having organs such as your skin, liver, kidneys and lungs, your cells on the micro level have organs, referred to as organelles with really neat names like the Golgi apparatus, endoplasmic reticulum and mitochondrion.  My favorite organelle is the mitochondrion.  Not just because it IS what keeps us going, but because it looks like a little maggot or blimp floating around in our cytoplasm.

The mitochondrion can be viewed, for the sake of analogy, as a parasite.  It has its own DNA, but doesn’t live on its own.  It has to live in our cells.  In return for a place to live the mitochondrion provides major benefits to its host – it is what makes every metabolic function in your body possible.  The mitochondrion is the “energy producer” that fuels all other metabolic functions.  Without these little parasites we would be toast.

Unlike nuclear DNA which is made up of alleles donated by mom and dad, the DNA in your mitochondria comes directly from your mother.  Doesn’t matter if you’re a man or a woman, your mitochondrial DNA will be determined by your mother.  This is why mitochondrial DNA testing, denoted by mtDNA, is used in genealogy a lot.  It helps you determine your line of heritage.  Mutations in mitochondrial DNA are very rare and therefore, you can have mtDNA testing done and the analysis will show the haplotype, also referred to as mitotype, you belong in.  A haplotype is basically a group of the human population that have similar mtDNA.  Over the course of human history rare events that have caused significant mutations in mtDNA have occurred, and that has created these various haplotypes.  These changes, or haplotypes, is what creates “the races”.  So barring some great, rare mutating event, your mtDNA should match your mother’s, which will match her mother’s, which will match her mother’s, and so on.

In mtDNA testing we step away from the allele study and we focus on the nucleotides that make up DNA.  As we all know, DNA is a double helix structure that can be imagined by taking an extension ladder and twisting it around its longitudinal axis.

In our DNA ladder the vertical supports are made up of alternating segments of deoxyribose sugar and phosphates.  It is considered the “backbone” of the DNA and, just like with our extension ladder, it is the connecting support between the rungs.  The rungs are made up of “bases”.  In fact, each rung is made up of two bases that are chemically attached to one another.  So on our DNA ladder each rung could actually be split into two separate parts – one part is one base, and the other part is another base.  The chemical reaction that holds our rungs together is an important point to understand because there are only a set number of bases, and they only “connect” in certain pairings.

The bases are split into two groups:  purines and pyrimidines.  Purines cannot connect to other purines and pyrimidines cannot connect to other pyrimidines.  It must always be a connection of a purine to a pyrimidine.  Why is this so?  Because purines are bigger than pyrimidines, so thinking about our ladder, if we had a rung made out of two purines, the ladder would have a “bulge” in it because you just stuck two bigger pieces together and that rung would be wider than the rest of the ladder.  Conversely, if you stuck to smaller pyrimidines together that rung would be narrower than the rest of the ladder and you’d have a goofy looking ladder for sure.  So, to keep the DNA ladder the same width along all of its length, you put a small pyrimidine with a big purine – always.

In addition to this requirement, only certain purines can hook up with certain pyrimidines.  This is due to the constraints of chemical reactions that will bind the two parts of our rungs together.  The purines are:

Adenine (denoted as A)

Guanine (denoted as G)

The pyrimidines are:

Cytosine (denoted as C)

Thymine (denoted as T)

The “base pairing” that is required is as follows:

A to T

G to C

So if we looked at every rung of our DNA ladder it would either be a A-T rung or a G-C rung…of course, these can be reversed so we could have a T-A rung and a C-G rung, but we will never have a A-C rung or a T-G rung.  It just can’t chemically happen.  Understanding this is important to understanding how mtDNA testing is achieved.

mtDNA testing involves the Polymerase Chain Reaction (PCR).  What PCR involves is pulling the two parts of our DNA ladder apart and using them to create, or amplify, the mtDNA sample.  So we can start with one little bitty piece of mtDNA and within two hours, using PCR, we can have a substantial sample to test.  To visualize how this is done, we will move from looking at the mtDNA as a ladder, and instead look at it as a little zipper.  So the first step in PCR is to “unzip” the two halves of the double helix.  Once we have separated these two halves we have the “negative”, if you will, of each other.  Remember, the base pairing must be an A to a T and a G to a C, so when we separate the two halves of the DNA zipper, we will have the two halves of the base pairing without its “mate”.

Remembering that chemically nothing else can attach to an A but a T, and nothing else but a C can attach to a G, so we can now take the two halves of our original piece of DNA and we can chemically build the other half of each side of the original DNA by just supplying the four bases and letting nature take its course.  In other words, if we supply enough A, T, G and C, they will find their mate on each half of the original zipper we unzipped and build two new pieces of DNA that are identical to the first piece of DNA we started with.  We’ve turned one DNA segment into two.  We continue to repeat this process…2 to 4, 4 to 8, 8 to 16, etc. and before long…we’ve got a lot of DNA to test.  From this we can return results on what the nucleotide sequence is for the sample DNA.  This result will be in the form of a position of the base being reported (say, position 16,126) and the base that is at that location (A,T,G or C).

In the results reported yesterday the first mtDNA data we have is a comparison of the mtDNA taken from the infamous Q12.1 hair retrieved from Casey’s trunk.  This is the single hair that shows signs of decomposition and a “death band”.  It is in this reporting that the FBI was able to conclude the hair was Caylee’s.

So what we have is that Casey’s mtDNA matches the mtDNA of the hair.  So how did they determine it was Caylee?  Well, remember, your mtDNA will be that of your mother, so the results of Casey apply to Caylee – i.e. Caylee’s results would be identical.  Since the mtDNA results of the hair could be Casey or Caylee, the determining factor that it was Caylee is the simple fact the hair came from a dead body, and Casey isn’t dead.  (A fact Casey apparently was proud to announce in front of Tracy McLaughlin.  According to Tracy they were watching news coverage of the forensics on the hair and when it was stated that the mtDNA matched both Caylee and Casey, but the hair was from a dead body, Tracy states Casey announced “Well, I’m still alive!”  Thank you, Casey, for helping us solve this one.)

After the nucleotide sequence is established for the sample DNA, it is compared to a reference standard.  The reference used in mtDNA testing is the Cambridge Reference Sequence (CRS).  It has been used since 1981.  The results from this comparison will be returned as a count of how many of the bases do not match the reference at particular locations.  So if you exactly match the CRS, the result will return “no differences” and your mtDNA is that of the CRS.  If you have differences, the returns will be reported as the location of the base, and what your base is.  Any location not reported is assumed to be a match to the CRS…so you only get the differences.  Here we have the results of the comparison of Q12.1 with the CRS (page 177).  The third column entitled “Con” is the sequence for the sample being tested – in this case the “death hair”.

So there are 8 locations where the base for Caylee/Casey differ from the CRS.  This can now be used to find their mitotype – i.e. their heritage.  Beginning on page 184 you can follow this process.  Starting on page 188 you can view where they are looking at the statistical analysis of the comparison of Caylee’s mtDNA with the various haplotype groups in their database.  To understand what is going on look at any given one of the group results and notice the first column says “Number of differences from search profile”.  The important row under this is “0″…that means you have a dead-balls match…there are no differences.  So as you step through the various haplotype groupings what you want to look for is the grouping where the highest number of perfect matches shows up.  This is found at the bottom of page 188 in the Caucasian Database.  There are 24 matches out of a total of 1,814 in the database for Caylee’s profile.

The other important number to look at is the “Upper Bound Frequency Estimate (%)”.  This is the number of people per 100 that fall into the Caucasian database profile and match Caylee’s mtDNA perfectly.  So for this result we find that 1.85 out of every 100 Caucasian people who fall into this haplotype will have the same returns as Caylee.  This is with 95% confidence.  While the mitotyping isn’t all that important to this case, I thought we’d at least go over it so you can understand what you are looking at.  In addition, it might be helpful to you if you ever decide to have mtDNA testing done for the purpose of lineage.

The next set of results are a comparison of the hair recovered from the label of Brian Burner’s shovel (Q46.2) and Casey’s mtDNA results.

If you’ll recall in previously released discovery, there were reports that stated the mitochondrial DNA evidence from the shovel did not match Casey or Caylee.  This is where it comes from.  Note that the hair from the shovel matches the CRS, but as we have already established, Casey has 8 differences compared to the CRS.  The hair, in fact, does have two differences, but is clearly not a match to Casey or Caylee’s mtDNA results.

And then the last set of mtDNA testing results we are given is the analysis on Q59.1 which was a hair taken from the hair mass found with the remains.  Here we see the comparison with Casey.

As can be seen, it is a match.  The comparison of the Q59.1 mtDNA sequences to the CRS return the exact same results as that done on Q12.1 (the death hair) and Casey.

Hence, not only do we have the nuclear DNA identification of the tibia for Caylee, we have the mtDNA identification of her hair via the comparison with Casey’s mtDNA.

Valhall.

Useful references:

http://www.blc.arizona.edu/Molecular_Graphics/DNA_Structure/DNA_Tutorial.HTML
http://www.mitochondrialdnatesting.com/
http://www.dnaheritage.com/mtdna.asp
http://www.familytreedna.com/tr_mtDNA.pdf
http://www.dfs.virginia.gov/manuals/forensicBiology/procedures/212-D100%20Forensic%20Biology%20Section%20Mitochondrial%20DNA%20Unit.pdf
http://www.dnalc.org/resources/animations/pcr.html
http://www.sumanasinc.com/webcontent/animations/content/pcr.html

Related posts:

1. Will Duct Tape be Casey’s Sticking Point? – The DNA
2. Understanding Forensics: Roles, Terminology, and the Null Hypothesis
3. Adipocere and DNA testing
4. Understanding Accumulated Degree Days
5. Making Sense of the Serological Results

Tags: casey anthony, caylee anthony, DNA, mitochondrial dna testing, mtDNA, polymerase chain reaction