Breaking News! Final Proof!
Here is a link to Dr. Coble's published journal of DNA results on the bones found in 2007, proving once and for all every member of the family died in 1918 and is now accounted for!
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004838
DNA and what it means
There have now been four different DNA tests involving the Anastasia question: The 1991 Romanov bones tested with blood from the royals, the1994 testing of AA's intestines vs. the pattern sequenced from blood of the royal family, the testing of AA's hair vs. the DNA of the royal family, and most recently, the testing of the charred bone fragments found in 2007 with the 1991 bones. The results prove to us that Anderson was not Anastasia, and that Anastasia died with her family in 1918. The questions and answers immediately following deal with the tests on Anderson's tissue samples and the likelihood she was Franziska:
What is DNA?
All living things have DNA, and those of us who are related share some or all of the same DNA. This is why by testing DNA, we can find out who is or is not related. This is what was done in the Anna Anderson case. Here is a link that describes and explains DNA:
http://ghr.nlm.nih.gov/handbook/basics/dna
Where did the DNA for Anna Anderson this test come from?
Upon her death, Anna Anderson, then called Anastasia Manahan, was cremated. This was her own decision. Some people believe she did this to prevent any tests from being done on her body after she was dead. But she had forgotten that a piece of her body was still around. In 1979, she underwent surgery for an intestinal problem at Martha Jefferson Hospital in Charlottesville, VA. The piece that was removed was safely cataloged and stored away. In the 1990's, when DNA testing became available, Richard and Marina Schweitzer, daughter and son in law of Anderson supporter Gleb Botkin, were so sure that she would be proven to be Anastasia that they pushed to find and test the body tissue. The sample was located, mtDNA was extracted, and tested against blood and hair samples from Prince Phillip, who besides being Alexandra's great nephew is the husband of Queen Elizabeth II. There was no match. Later, a hair sample belonging to Anderson was found in an old book and tested as well. Again, no match to the royal family. In addition to not matching the royal family, Anderson's DNA was a perfect match with Karl Maucher, great nephew of Franziska Schanzkowska, the Polish factory worker who went missing in the same time frame Anderson appeared, and who was identified by detectives in the 1920's as Anderson's most likely true identity. Anna Anderson was Franziska Schanzkowska, NOT Anastasia.
Couldn't the sample have gotten switched or mixed up at the hospital?
This is the explaination of Penny Jenkins, who was responsible for Martha Jefferson Hospital's medical records, including blood and tissue samples. When asked of the possibility of 'substitution' of the tissue at the hospital, here was her reply:
"We have two separate backups. In 1979 when Dr. Shrum did surgery on Mrs. Manahan, we took slides of the tissue, in addition to preserving in paraffin the larger blocks of the excised tissue. Taking slides when doing surgery is routine, you take it, you look at it, and say, there is cancer, or it's not cancer, or it's an infection or whatever. We preserve these slides in one place and the paraffin wax in a totally different place.
"Furthermore, when we moved the tissue from storage back to the hospital in early 1993, Dr, Thomas Dudley, the assistant pathologist, cut some new slides from one of the blocks. We compared these new slides cut in 1993 with those slides cut in 1979 and they were identical. If someone had swapped them in storage during the last couple of years, they would not have matched. And the chance that anybody was able to get to both locations and switch both slides without access to specimen numbers is impossible."
(source: Massie, "The Romanovs: The Final Chapter")
What about how the hospital first claimed they had nothing in her name, then found it later. Isn't that suspicious?
No, the reason the hospital didn't find it right away was mainly because the person who originally asked for the sample gave the names "Anna Anderson" and "Mrs. Jack Manahan", but the sample was filed under the name "Anastasia Manahan" (her husband's real name was John, Jack was only a nickname, and it was under her name, not his, so this is why the computer searches originally gave no matches.)
(source: Klier and Mingay, "Quest for Anastasia")
For much more on the story of the legalities of obtaining the sample for testing, please see the lengthy information in "The Romanovs: The Final Chapter" by Robert K. Massie
How do we know they didn't find out what her DNA was and then make sure it didn't match?
Because Prince Phillip's mtDNA was sequenced ahead of AA's. Regardless, to no one's surprise, his mtDNA exactly matched that of Princess Katherine's and neither matched AA's.
The first Victorian mtDNA to be sequenced was not the Duke of Edinburgh's(Prince Phillip's), it was Princess Katherine of Yugoslavia's (Mrs. De Silva). The sample was taken in London and sent to Brown University where it was sequenced and compared to a purported offspring of Grand Duchess Tatiana Nicholievna. There was no match. Because a sequencing of the Victorian DNA already existed and was documented, there was no way it could have been faked or invented just to trick AA supporters as some claim.
But couldn't there have been some mistake? Aren't there some recent reports of errors in some tests?
In the days when the AA testing was done, there were only a few people who knew how to do it. They were the pioneers, and specialists who were very good at their work. The main scientists involved are still working today and have very highly regarded reputations in the scientific and legal communities. These days, there are a lot more DNA labs out there, and there are thousands of people working in them, some who are not as expert as they should be. There are even commercials inviting you to go to lab x downtown and get a DNA sample done of a hundred dollars. You can even have your dog's DNA tested. With all these new labs, some with questionable employees and procedures, there may be a mistake once in awhile. BUT the thing to consider and realize in the AA case is that, in addition to the high level of expertise and integrity of those involved, the tests were done in FOUR different labs- three for the intestines, one for the hair. All labs reached the same exact result. If one had 'goofed', one would not match the others. If they all goofed, none would match. It's impossible that they would all make the same random error. The tests were accurate, and the scientists involved will be the first to tell you there is no need to test them again, you will get the same result. If you have the money and the time and want to try to challenge it in court, go for it, but you will lose.
Now that the new DNA tests have been done on the entire family after the 2007 discovery of the two missing bodies, the 1994 tests have been shown to be more valid than ever, and the new tests have put an end to all doubt.
| Why do people still question the tests? |
|
Here is an analysis by a scientist who tries to explain just how high the probability is, and why:
What are the chances that Anna Anderson was Franziska Schanskowksa based on DNA?
By DaveK
Some AA proponents assert that AA’s specific mtDNA type is very common type, therefore a match between AA and FS is just by accident. However, this argument is fundamentally flawed. If so, why don’t they just show the data of someone who has same mtDNA? There are more than dozens populaiton genetics papers that you can check very easily. They can’t, because their claim is not true.
Before showing the evidence, I have to point out that the probability 1/300 reported in Peter Gill’s study in 1995 was outdated. Gill “guessed” the number from statistical average because he didn’t find AA’s mtDNA type in database available in 1995. Therefore, any unknown mtDNA in 1995 was estimated as “1/300” temporally, even if its actual probability is 1/5000 or 1/100,000 (!).
To get more accurate estimate, I checked all mtDNA (HVI) database available to me that contained 8,902 sequences of European Caucasian including US Caucasian, British, French, German, Italian, Spanish, Polish, Russian, Hungarian, Austrian, Dutch, Norwegian, Swedish, Ashkenazic Jewish, Belgian, Icelandic, Austrian, Bulgarian, Portuguese and so on. I also checked African and Asian population just in case. Most convenient sources are major human genetics journals such as Annals of Human Genetics and American Journal of Human Genetics (especially Annals of Human Genetics vol 67 (2003), p281 was helpful). Also computerized database were used, such as NCBI GenBank, European Molecular Biology Laboratory (EMBL), and US Department of Justice FBI CODIS database.
The reason why I investigated different regions separately was to see “population structure” due to ethnic subgroup, but prevalence of Tara clan was 10 +/- 2% in all countries in Europe, which indicates there is no siginificant structure (also see Science Vol 254 p1735). I’ll discuss this issue in Question 3.
TABLE 4 (Some examples of European mtDNA (HVI) studies)
---------------------------------------------------------------------
French (total = 109)
9 person has the most common type: CRS (no mutation)
Almost all other 93 person has a unique mtDNA (does not share mtDNA each other).
No one has AA’s mtDNA (16126C, 16266T, 16294T, 16304C)
----------------------------------------------------------------------
Autstrian (total = 101)
9 person has the most common type: CRS (no mutation)
Almost all other 80 person has a unique mtDNA (does not share mtDNA each other).
No one has AA’s mtDNA
----------------------------------------------------------------------
British (total = 100)
12 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
-----------------------------------------------------------------------
Russians and Ukrainians (total = 201)
22 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
-----------------------------------------------------------------------
Polish (total = 436)
67 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
-----------------------------------------------------------------------
US Caucasians total = 323
61 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
In all regions, by far the most common mtDNA haplotype (HVI) is CRS (Cambridge Reference sequence). About 10% of population in any country (except US) has this sequence (almost same prevalence as AB blood type), i.e. about 65 million European has an exactly same mtDNA sequence (at HVI). There is no known reason why this specific type is so prevalent. It seems just stochastic genetic drift event. A friend of mine jokes this mtDNA type is related to “beauty phenotype” expressed in their daughters, but I don’t think it’s true. (By the way, this CRS sequence itself from a British woman whose identity kept secret for some reason since 1981. A rumor goes that it was a researcher’s wife’s mtDNA.)
However, this CRS mtDNA is an exception. Almost all other mtDNA type is rare, usually less than 1%. For example, I checked Tsarina’s mtDNA type 16111T/16357C. There was 0 in database of 8902 caucasians. Tsar’s mtDNA was also rare, 0 out of 8902. And Anna Anderson’s mtDNA had 1 in 8902 (1 found in Iceland study). therefore the random match probability is 1/8902 = 0.01%: about 30 times rarer than the original Peter Gill’s estimate (1/300).
So, can I conclude from this DNA evidence alone? Not so fast. I think many people confuse DNA’s random match probability, likelihood ratio, with Posterior Odds. To discuss if AA is FS, we have to discuss posterior odds.
Bayesian inference is the logical/mathematical framework to interpret the combined probability of independent event. Forensic science in both US and UK are always interepreted in a logical sturucture of Bayesian inference. In the court, forensic exprert are instructed by judge to testify only regarding to “DNA random match probability” or “likelihood ratio”, but what really concern jury is the posterior odds. Here I try to be a jury rather than a DNA expert.
O (posterior) = O (prior) * DNA likelihood ratio
Roughly speaking, if two person’s sex, age, physical feature including height, hair color, face feature, prior odds are 1:10. Considering FS has been missing at almost exactly same time at same geological area as AA appeared, even conservative odds brings this to 1:100. DNA random probability is a simply inverse of likelihood ratio in this case, so my calculation shows:
O (posterior) = 1/100 x 1/9000 = 1/900,000 (that is to say, probability that AA is FS is 99.9999%)
As “reasonable doubt” is generally considered O(posterior)(threshold)
=1/10,000, it is reasonable to accept hypothesis that “AA is FS”.
Therefore, with overwhelming evidential support and lack of alternative scenario, I support the hypothesis that AA= FS.
Anna Anderson was FS = 99.9999%
Anna Anderson was Anastasia = 0.00000000 (add 80 of zero here)0001% *
FS was murdered by Grossmann = 0.00001%
FS was murdered by other murders = 0.00002%
FS was killed by accident = 0.00002%
FS was living peacefully under other pseudonym = 0.00004%
FS was kidnapped by foreign intelligence agency such as KGB = 0.00001%
FS didn’t exist from beginning, she was a fiction by her family= 0.000001%
Anna Anderson was Franzkiska Schanzkowska = 99.9999%
But aren't the 1994 tests now outdated? Don't they need to retest using the newer 23 point testing?
Not in the case of mtDNA, or the Anna Anderson tests.
Perhaps this explaination will help.
For example (this is going to be extremely oversimplified, but ok for our purposes). Here are two sequences:
ACTGGGTAACGTAAGGTC
AGTAAGCCACTATACGCC
So we are comparing them to see if the two match. Normally you won't compare the entire sequence but just part of it...
So if we look at random loci (positions) then there is a chance we may get a false positive if we don't look at enough of them... Like this:
ACTGGGTAACGTAAGGTC
AGTAAGCCACTATACGCC
So even though these specific loci match, the sequence doesn't. Statistically speaking, the more loci you compare the more accurate the result will be, because in this case, if you did one more, there would be a mismatch and we would have our answer.
However, with mismatch, it's a different story once there is a mismatch, even of one base. So if you look at the same sequence but compare more loci and get one mismatch, it's a mismatch, period. There is no way you can a false mismatch... Like this:
ACTGGGTAACGTAAGGTC
AGTAAGCCACTATACGCC
And once there is a mismatch, even ONE mismatch, all bets are off.
To be clear. Whether one looks at 13 or 23 loci, ONE mismatch is an exclusion for mtDNA. The case of Anna Manahan had FIVE mismatches to the Victoria line of descent. There were NO mismatches for the Karl Maucher mtDNA. IF one examines 23 loci, the same five mis-matches will STILL be there. This is the reason that every single specialist in forensic mtDNA analysis says there is no reason to re test the Anna manahan samples.
Yes the entire genome is vast, BUT every single human being shares the exact same sequences over 99.5% of the genome. The actual amount of variation is rather small, and occurs in what is called "junk DNA". Only certain strings of junk DNA will match with close blood relations. HOWEVER, only two or three max. mis matches will exclude relationship 100% no doubt. THIS is why those claiming the mtDNA of Anna Anderson should be retested or is "unrealiable" are simply ignorant, misguided or deliberately avoiding the reality.
Thank you for sitting through science class! The hypothesis has been proven, the conclusion is evident.
Anna Anderson was Franzkiska Schanzkowska = 99.9999%
More explaination on why and how AA was FS
Anna Anderson’s DNA and population genetics
By DaveK
In 1995, British forensic scientist Peter Gill published an article in a journal Nature Genetics that claimed that DNA from Anna Anderson (AA) did not match that of Romanov, but matched a great nephew of a polish worker Franziska Schanzkowska (FS). Peter Gill, an internationally renowned forensic expert who pioneered both mtDNA- and STR-based DNA profiling technologies, concluded that the random chance for matching two is less than 1/300.
However, this physical evidence did not convince all. The study has aroused strong opposition from pro-AA believers, a small but passionate group who supports the view of an influential author Peter Kurth (*1). The AA proponents are endlessly creative, generating a new theory which challenges the validity of the DNA result every year. By no means should I expect the defender of AA to show uncritical acceptance of the scientific study. However, all criticisms from pro-AA so far are rife with factual errors and fallacious argument often with the deceptive rhetoric device of pseudoscience.
The purpose of this post is to offer scientific clarification to this issue by using the recent population genetics data obtained during past ten years, many of which were not available in 1995. For example, seven main phylogenetic lineages of mtDNA among European caucasian were not clearly identified in 1995. I will concern five questions:
1) Who is AA?
2) Is it true that AA’s mtDNA is actually much more prevalent than 1/300?
3) Because of the rapid evolution of forensic technology, will the “old” method Peter Gill used become invalid in the future?
4) Was DNA possiblily contaminated?
5) and Why all these reasoning never convince pro-AA believers.
Before tackling the serious issues, I will try to answer one pseudo-question
Q1) “Who is AA? Is AA possibly a distant relative of Tsar?”,
Because this question help us to understand how the population genetics work.
Unlike the fingerprint, DNA contains “memory” of your genealogy information. Just like a name “Jacqueline Lee Bouvier Kennedy Onassis” can tell you her family history, the profile of mtDNA can tell you information of your ancestor, to a certain degree. Let’s use my own mtDNA as an example (you can get your own for $200). Mine has a G mutation at the site 16162 (i.e. 16162G). Table 1 shows that I belong to Helena clan (*2). I am a descendant of Helena, a Caucasian woman existed in Euproe 20,000 years ago, and her other descendant include Queen Victoria, Tsarina Alexandra, and Marie Antoinette.
TABLE 1------------------------------------------------------------------------------------------
Seven European mtDNA Haplogroups (or clan)
Haplogroup: Clan mutation in HVI population (%)
H: Helena (no change) 46%
U: Ursula 16270 21%
J: Jasmine 16069+16126 9%
T: Tara 16126+16294 9%
K: Katrine 16224+16311 6%
V: Velda 16298 4%
X: Xenia 16223 1%
(*Note) Douglas Wallace assigned seven letters to seven haplogroup, but an entrepreneur Bryan Sykes begun to use more familiar term “seven daughters (clan) of mitochondria Eve”. I try to use daughters’ names for non-scientists to understand them easily.
TABLE 2------------------------------------------------------------------------------------------
MtDNA haplotype of Romanov family and others
Sample HVI Seq Haplogroup: Clan mother
Cambridge Reference Seq HVI* CTGCCCCCACCTTCT H: Helena
Anna Anderson +C++++T++T+C+++ T: Tara
G nephew of FS +C++++T++T+C+++ T: Tara
Tsar Nicholas II +C+Y+++++TT++++ T: Tara
Servant 1 +++++++++++C+++ H: Helena
Servant 2 ++++++++++++C++ H: Helena
Servant 3 ++++T++TG+++C++ H: Helena
Dr Botkin (putative) +++++T+++++++++ H: Helena
Romanov Daughter 1 T+++++++++++++C H: Helena
Tsarina Alexandra T+++++++++++++C H: Helena
Prince Philip T+++++++++++++C H: Helena
Marie Antoinette T+++++++++++++C H: Helena
(*15 positions in HVI are shown: 16111, 126, 129, 169, 261, 264, 266, 278, 293, 294, 296, 304, 311, 327, 357)
(“+” indicates no difference from Cambridge Reference)
AA’s mtDNA is 16126C, 16266T, 16294T, 16304C
Tsar’s mtDNA is 16126 C, 16169Y, 16294T, 16296T
(Note: * By the way, the fact all mtDNA from Gill’s study in 1994 matches with the Seven Daughers profile is important. Because if Gill’s group had “faked” the result it should show some inconsistency, because there was no way to predict “correct” Caucasian mtDNA type.)
TABLE 3------------------------------------------------------------------------------------------
STR loci, Tsar’s genotype, Anna Anderson’s genotype
VWA--------- (15, 16) (14, 16)
TH01--------- (7, 9.3b) (7, 9.3)(*3)
F13A1-------- (7, 7) (3.2, 7)
FES/FPS----- (12,12) (11, 12)
ACTBP2----- (11, 32) (15, 18 )
Let’s take a look at Romanov family’s mtDNA (Table2). Obvisouly Anna Anderson is not Tsarina’s daughter, but her mtDNA is similar to Tsar. Two out of four mutations are at exactly same position. Not only this, if you look at table 3, she shares four out five STR mutation with Tsar. Does it mean that Anna Anderson is a distant relative of Tsar? Some witness claimed striking similarity of almost mystic “atmosphere” of Anna Anderson to Anastasia. What if they are second cousin or something?
Thanks to data that was collected for past ten years, we can answer this question. Both Tsar and Anna Anderson has a same ancestor Tara, who was one of seven daughters of Eve, who existed about 36,000 years ago. From this starting point, any mutations could occur in a stochastic fashion. If four new mutations which were not found in Tara occurred very very recently, Tsar and AA could be a relative each other. Is that so?
Unfortunately, genetic evidence doesn’t say so. All the data shows that these four mutations happened long time ago, at least 10,000-30,000 years ago. This can be determined by the chronological order of mutation event. By drawing phylogenetic tree, the order of mutation in AA’s mtDNA was detemined and it’s 16126>16294>16304>16266. One Russian population study found that there is a few descendant clan of AA’s clan whose mtDNA is 16111, 16126, 16294, 16304, 16311, 16327,16266, i.e., 3 mutations in addition to AA’s 4 mutations, which indicates AA’s 4 mutations happened long time ago. So if we take the position that mutation occurred in a semi-linear fashion to a first approximation, which means every 12000 years, the following is likely:
36,000 years ago----Tara
24,000 years ago----Mutation 16304 occured in AA’s ancestor. Mutation 16296 in Tsar’s.
12,000 years ago----Mutation 16266 occurred in AA’s ancestor. Mutation 16169 in Tsar’s.
In conclusion, (from the viewpoint of maternal lineage) AA and Tsar had a same ancestor, but it was about 36,000 years ago. That is to say, their great great great great great great great great ……(repeat 1800 times!!)…… great great great grandmother was a same woman.
--------Footnote-------------------------------------------------------- ----------------------
*1, Peter Kurth still believes AA is Anastasia, but he does not employ pseudoscience tactics to convince other people. He simply say “I just don’t trust DNA".
*2, it may be confusing. The table shows only 15 mutations, but it doesn’t show my mutation 16162. It means that I belong to Helena clan.
*3, Original Peter Gill’s paper shows (7, 9, 3b) as Tsar’s genotype, but it’s a typo of (7, 9.3b) as there is no (7.9) genotype.
Question 2) Is the random match probability of AA’s DNA really 1/300?
Some AA proponents assert that AA’s specific mtDNA type is very common type, therefore a match between AA and FS is just by accident. However, this argument is fundamentally flawed. If so, why don’t they just show the data of someone who has same mtDNA? There are more than dozens populaiton genetics papers that you can check very easily. They can’t, because their claim is not true.
Before showing the evidence, I have to point out that the probability 1/300 reported in Peter Gill’s study in 1995 was outdated. Gill “guessed” the number from statistical average because he didn’t find AA’s mtDNA type in database available in 1995. Therefore, any unknown mtDNA in 1995 was estimated as “1/300” temporally, even if its actual probability is 1/5000 or 1/100,000 (!).
To get more accurate estimate, I checked all mtDNA (HVI) database available to me that contained 8,902 sequences of European Caucasian including US Caucasian, British, French, German, Italian, Spanish, Polish, Russian, Hungarian, Austrian, Dutch, Norwegian, Swedish, Ashkenazic Jewish, Belgian, Icelandic, Austrian, Bulgarian, Portuguese and so on. I also checked African and Asian population just in case. Most convenient sources are major human genetics journals such as Annals of Human Genetics and American Journal of Human Genetics (especially Annals of Human Genetics vol 67 (2003), p281 was helpful). Also computerized database were used, such as NCBI GenBank, European Molecular Biology Laboratory (EMBL), and US Department of Justice FBI CODIS database.
The reason why I investigated different regions separately was to see “population structure” due to ethnic subgroup, but prevalence of Tara clan was 10 +/- 2% in all countries in Europe, which indicates there is no siginificant structure (also see Science Vol 254 p1735). I’ll discuss this issue in Question 3.
TABLE 4 (Some examples of European mtDNA (HVI) studies)
---------------------------------------------------------------------
French (total = 109)
9 person has the most common type: CRS (no mutation)
Almost all other 93 person has a unique mtDNA (does not share mtDNA each other).
No one has AA’s mtDNA (16126C, 16266T, 16294T, 16304C)
----------------------------------------------------------------------
Autstrian (total = 101)
9 person has the most common type: CRS (no mutation)
Almost all other 80 person has a unique mtDNA (does not share mtDNA each other).
No one has AA’s mtDNA
----------------------------------------------------------------------
British (total = 100)
12 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
-----------------------------------------------------------------------
Russians and Ukrainians (total = 201)
22 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
-----------------------------------------------------------------------
Polish (total = 436)
67 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
-----------------------------------------------------------------------
US Caucasians total = 323
61 person has the most common type: CRS (no mutation)
No one has AA’s mtDNA
In all regions, by far the most common mtDNA haplotype (HVI) is CRS (Cambridge Reference sequence). About 10% of population in any country (except US) has this sequence (almost same prevalence as AB blood type), i.e. about 65 million European has an exactly same mtDNA sequence (at HVI). There is no known reason why this specific type is so prevalent. It seems just stochastic genetic drift event. A friend of mine jokes this mtDNA type is related to “beauty phenotype” expressed in their daughters, but I don’t think it’s true. (By the way, this CRS sequence itself from a British woman whose identity kept secret for some reason since 1981. A rumor goes that it was a researcher’s wife’s mtDNA.)
However, this CRS mtDNA is an exception. Almost all other mtDNA type is rare, usually less than 1%. For example, I checked Tsarina’s mtDNA type 16111T/16357C. There was 0 in database of 8902 caucasians. Tsar’s mtDNA was also rare, 0 out of 8902. And Anna Anderson’s mtDNA had 1 in 8902 (1 found in Iceland study). therefore the random match probability is 1/8902 = 0.01%: about 30 times rarer than the original Peter Gill’s estimate (1/300).
So, can I conclude from this DNA evidence alone? Not so fast. I think many people confuse DNA’s random match probability, likelihood ratio, with Posterior Odds. To discuss if AA is FS, we have to discuss posterior odds.
Bayesian inference is the logical/mathematical framework to interpret the combined probability of independent event. Forensic science in both US and UK are always interepreted in a logical sturucture of Bayesian inference. In the court, forensic exprert are instructed by judge to testify only regarding to “DNA random match probability” or “likelihood ratio”, but what really concern jury is the posterior odds. Here I try to be a jury rather than a DNA expert.
O (posterior) = O (prior) * DNA likelihood ratio
Roughly speaking, if two person’s sex, age, physical feature including height, hair color, face feature, prior odds are 1:10. Considering FS has been missing at almost exactly same time at same geological area as AA appeared, even conservative odds brings this to 1:100. DNA random probability is a simply inverse of likelihood ratio in this case, so my calculation shows:
O (posterior) = 1/100 x 1/9000 = 1/900,000 (that is to say, probability that AA is FS is 99.9999%)
As “reasonable doubt” is generally considered O(posterior)(threshold)
=1/10,000, it is reasonable to accept hypothesis that “AA is FS”.
Therefore, with overwhelming evidential support and lack of alternative scenario, I support the hypothesis that AA= FS.
Question 3) Because of the rapid technical evolution, will the “old” method Peter Gill used become invalid in the future?
First of all, becoming "obsolete" and becoming "invalid" are two different issues. Any technology becomes obsolete eventually. Since the DNA Identification Act of 1994, US government spent significant amount of our tax dollars for Department of Justice to advance a forensic technology every year. In fact, FBI is now developing a “real” DNA profiling technology which can determine suspect’s hair color, eye color, height, and even facial virtual montage from crime scene DNA (As of 2004, only “red hair” gene was isolated). In the future (at least a decade), chance are that a scientist could generate a virtual montage picture of FS (?) from AA’s DNA, or daughters picture from Romanov bones.
Even if the forensic technology advances, all data shown in Gill’s article is rock-solid today. Using table 5, I explain two aspects of the STR technique: STR and number of probes.
TABLE 5------------------------------------------------------------------------------------------
Timeline of main event in forensic technology
1986 -----First use of DNA in criminal case
1987 -----Castro case raised concern among forensic experts, prompting to establish a standard guideline and national database.
1988 -----First PCR combined with dot blot hybridization used
1991 -----First STR (4 multiplex STR) developed in UK.
1992 -----NRC report I, establishing controversial “ceiling principle”.
1992 -----First forensic use of mtDNA PCR
1994 -----Second generation STR (6 multiplex STR) developed in UK.
1994------First use of STR-PCR for anthological study by Peter Gill
1995 -----Peter Gill published the result of AA’s DNA.
1996 -----NRC report II announced in US, rejecting “ceiling principle”.
1997 -----US FBI announced the CODIS using 13 multiplex STR (match probability 10E-15).
2000 -----UK added 4 loci to STR, reducing the match probability to 10E-13.
2004 -----UK and US’s national DNA database’s profile size reached 2.5 millions, and 1.5 millions, respectively.
First, It is true that DNA profiling in early development phase was not free from mistake. Notorious case was Castro case, when there was no objective guideline to interpret dot blot hybridization result, creating subjective bias in data evaluation. Another problem in earlier phase was “binning approach” using polymorphic VNTR. However, this is the very reason why Peter Gill developed multiplex STR, which is faster, better, and more accurate. He did not use any dot blot or binning approach in his paper. So this point is irreverent.
Secondly, there was a huge controversy regarding “ceiling principle” mandated by National Research council (NRC) repot I in 1992. This ceiling principle which overestimated population structure (ethnic group) was criticized sharply because it rationale was not academically founded and it produced the statistical result that is abnormally conservative. Because of the controversy, ceiling principle was abandoned in a new NCR repot II in 1996. Consensus among population geneticist was that although population structure does exists, its effect was negligible in practice. This point is irreverent to Gill's study. It was essentially irreverent to my answer two above, but just in case I considered population structure (and even substructure) by studying mtDNA type by each country, and there was no significant structure observed.
Thirdly, it is true that numbers of STR multiplx kept increasing from 4 STR to current FBI CODIS’s 13 STR. Logical fallacy that pro-AA believers employ is that 4 STR is less “accurate” than 13 STR. In fact, number of STR does not affect “accuracy” of each probe. The reason why the authority increased the STR multiplex is following:
(1) to perform screening process against DNA database which contains millions of potential suspect. In earlier day, this was not the issue, because database itself did not exist. And the main purpose of STR was not individualization of suspect, but to exculpate the innocent. Remember, the first phase of paternal test of Anna Anderson was also a exculpatory (to prove she was not daughters of Romanov)
(2) to distinguish the suspect from unknown brothers/sisters so that it can withstand the most rigorous challenge from defense attorney. Imagine the attorney challenges DNA expert like this: “What if the suspect has a brother or sister who we don’t even know exists, and what if he/she committed the crime? As brothers have very similar DNA, is it possible that the DNA belongs to them?” Using 13 STR, which discriminate 600 trillions people, expert can now say “No, even if brother exists, it’s not him.” But obviously this special case does not concern AA case.
Even if it’s not exculpatory, STRs Gill used in the paper was still extremely rigorous. Table 6 is the calculation of allele frequency of Anna Andersons’ DNA:
TABLE 6------------------------------------------------------------------------------------------
VWA,------- (14, 16) 0.125 x 0.22
TH01,------- (7, 9.3)(*3) 0.145 x 0.3080
F13A1------- (3.2, 7) 0.032 x 0.424
FES/FPS -----(11, 12) 0.4030 x 0.2450
ACTBP2 -----(15, 18 ) 0.037 x 0.0705
Amelogenin -----(X, X) 0.5
P = 0.125 x 0.22 x 0.145 x 0.3080 x 0.032 x 0.424 x 0.4030 x 0.2450 x 0.037 x 0.0705 = 0.0000000021.
The discrimination power of this STR is one in half billion. As population of Europe is about 600 millions, it is pretty high. Remember this was the first exculpatory test to “screen” the matching possibility. And if Gill had match to parents (Tsar and Tsarina), he could have done the second phase of individualization using more STR. In fact, performing 20 STR for this first phase would be not only necessary, but inappropriate. If Gill performed 20 STR test, peer reviewer would have begun to suspect if there was something wrong with the whole procedure. There is no reason to waste a precious anthropological sample.
What is most mysterious to me is that the handwriting or ear-mark (from old photo!) evidence that pro-AA believers worship has a overwhelmingly low power of discrimination. Both type of tests’s resolution is only 0.1-0.01. If they claim such evidences are reliable, how could they criticize 4-multiplex STR?
Question 4) Is contamination possible?
I agree that all PCR-based DNA result should be subject to rigorous scrutiny because of potential contamination. However, the ambiguous word “contatmination” generates misunderstanding.
For example, if you are trying to find dinosaur’s DNA from fossil, and if the sequenced result is similar to human, the contamination will be very likely, because human DNA is ABUNDANT in our environment. However, if you are working with human sample, you don’t have to worry about contamination of dinosaur’s DNA. This is called “directionality principle of contamination”, that is to say that contamination happens in an asymmetric way, only from abundant source of A to rare source of B (or if amont are almost equal, it’s bidirectinal).
Anna Anderson’s DNA was obtained from four samples, including one intestine sample and three hair samples. If all DNAs had shown the most common human mtDNA type (CRS), then the possibility of contamination would not be excluded so easily. But that is not the case.
Her mtDNA was very rare (1/8902). The chance of this contamination happening was:
p = 1/8902 x 1/8902 x 1/8902 x 1/8902 = 0.000000000000000000000016 (1 in 10,000 trillions). Therefore, with overwhelming evidential support and lack of alternative scenario, I support the hypothesis that contamination is extremely unlikely.
What about deliberate contamination, or conspiracy theory? Besides its violation of falsification principle, this theory violates physical law. Non-medical people misses an important point in the Gill’s paper. Anna Anderson’s DNA was from the formaldehyde fixed sample of intestine tissue. Our current biomedical technology has no way to generate intestine tissue in vitro, even if you use any type of stem cell-related regeneration or organ cloning technology.
Only way to fake this sample is to abduct one of AA’s maternal relative (!) and do surgery to remove intestine from their stomach while they are unconscious, which is as unlikely as alien abduction. Adding synthesized DNA is also impossible. Many pro-DNA believers don’t know the fact that there are so many possible DNA profiling tests (more than 200 STR, and many different VNTR, mtDNA, and all others STRs along three billions nucleotides of human DNA) that it is impossible to predict what test the scientist will perform. Also from chemical properties (methylation pattern, pyrimidine dimers etc), any synthesized DNA can be distinguished from endogenous DNA easily.
My conclusion: conspiracy is not possible.
Question 5) Why don't they accept the hypothesis based on evidence?
Finally, the most important question: Why do they believe such an absurd theory?: I am sure none of my conclusion above doesn’t shake the belief of AA proponent. You may regard them as almost pathologically gullible. I don’t think that’s the case. For past twenty years, many psychologists studied the belief system of brain of those who believe ESP, UFO, creationism, homeopathy, fortune teller, astrology, hypnosis, Freudian psychoanalysis and so on. What surprised psychologists is that most believers are actually very normal people; sane, sober, honest, educated, and intelligent. Then what makes a difference?
According to psychologist James Alcock, our brain does not believe something because of its rationality. Rather, new belief is generated only when it is compatible with pre-established old belief. In addition, belief was solidified when data was combined with sensory information. As all sensory information go through the part of brain called amygdala, where emotions (fear, anger, joy, love, sympathy) affect incoming information dramatically, and our intellectual part of brain cannot control this process. This is like we fear trip by air plane even if we know it’s statistically safer than car.
If someone who was convinced by skilled writer like Peter Kurth because of its emotional impact towards “poor” Anna Anderson, any new information presented by some “esoteric” DNA evidence by abstruse scientist would have no chance to compete with the previous strong belief. As a result, they will forever seek for new “truth” until it match their belief, instead of matching truth to their belief.
I will welcome any refutation to my hypothesis, as long as it has an alternative scenario based on verifiable evidence which is at least as plausible as mine.
Gill Paper
Here is a section of the Gill paper, Nature Genetics,1995, so called "AA paper".
Gill P, Kimpton C, Aliston-Greiner R, Sullivan K, Stoneking M, Melton T, Nott J, Barritt S, Roby R, Holland M, et al.
Establishing the identity of Anna Anderson Manahan.
http://www.nature.com/ng/journal/v9/n1/abs/ng0195-9.html
http://img156.exs.cx/img156/9286/aaarticle4pv.jpg
A year later, in 1996, Nature Genetics declared the case closed (AA=FS end of story)
Nature Genetics. 1995 Jan;9(1):9-10.
Peter Gill, 13th INTERPOL Forensic Science Symposium, Lyon, France, October 16-19 2001:
MtDNA has proven invaluable in solving historical mysteries such as the fate of the Romanov family (Gill et al 1994) and testing the claim of Anna Anderson to be the Duchess Anastasia (Gill et al 1995). Dr. Gill calls the results invaluable to solving the mystery.
]Mitochondrial Steve: paternal inheritance of mitochondria in humans
Lindell Bromham, Adam Eyre-Walker, Noel H. Smith and John Maynard Smith
Centre for the Study of Evolution, School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex BN1 9QG, UK
mtDNA analysis has also been used to counter ongoing claims to the Romanov dynasty. In 1995, sequencing of mtDNA from Anna Anderson
Manahan showed that she could not have been Anastasia, daughter ofTsar Nicolas II, as she claimed [c].
References
c Stoneking, M. et al. (1995) Establishing the identity of Anna Anderson Manahan. Nat. Genet. 9, 9–10
NATURE REVIEWS, GENETICS, VOLUME 5
OCTOBER 2004
pgs 731 et seq
ENCODED EVIDENCE:DNA IN FORENSIC ANALYSIS
MarkA.Jobling and Peter Gill
The remains of one of the Tsar’s daughters,Anastasia,were absent from the grave,and controversy surrounded the
claim that she escaped execution and survived,under the identity of Anna Anderson.STR analysis of 20-year-old
paraffin wax embedded samples from Anderson was inconsistent with her being a daughter of the Tsar and Tsarina.
However,the mtDNA sequences matched those of Carl Maucher,a putative maternal relative of a woman named
Franzisca Schankowska.The mtDNA results were confirmed by an independent group from Penn State University,who
concurrently analysed hair shafts purported to have come from Anna Anderson
note131
131. Gill, P. et al. Establishing the identity of Anna Anderson Manahan. Nature Genet. 9, 9–10 (1995).
Schweitzer RR.
Anastasia and Anna Anderson.
Nature Genetics. 1995 Apr;9(4):345. 
Here is Gill’s original data from (Nature Genetics. 1995 Jan;9(1):9-10.) .
---------------STR profile of nuclear DNA from Anna Anderson--------------------
---------------mitochondria DNA profile from Anna Anderson--------------------
---------------mitochondrial DNA figure--------------------
---------------mitochondrial DNA figure--------------------
Schweitzer is making a bold statement in the letter.
1) Bernd Hermann's testing showed that DNA was completely different from the Peter Gill's result.
Here is my response: WHERE IS THE DATA? If this is true, why doesn't he show the different mtDNA profile? Schweitzer must know it, because he can't claim this unless he knows the sequence. And if he knew it, why didn’t he show the difference?
And why is Hermann silent while Gill and Nature make a false statement?
If anyone knows about this, please let me know.
2) He also claimed that Stoneking didn't get STR from hair sample.
My response: Peter Gill already showed that he couldn't get STR from old hair sample. It is difficult, if not impossible, to get the nuclear DNA from old hair, that's why they needed intestine sample. That’s the whole point of the paper.
3) Many points suggested by Schweitzer are a typical red herring. For example, he claimed that sex of the mtDNA source was not determined. Of course you can’t determine the sex from mtDNA of hair sample, but why does it matter for the argument for the matching between Anna Anderson and FS’s maternal relative?
4) This letter was written 10 years ago. He claimed he was going continuing his investigation. If he had any solid evidence to contradict the Gill’s result, why didn’t he publish (journal, book or even on internet) any of the result? Other critics of DNA testing like Alec Knight claims he will publish his new finding in a near future, but I am suspicious if they ever do it.
Here are the tables from Gill’s paper in 1994. It shows DNA profiles of nine skeletons.
Gill P, Ivanov PL, Kimpton C, Piercy R, Benson N, Tully G, Evett I, Hagelberg E, Sullivan K. Identification of the remains of the Romanov family by DNA analysis.
Nature Genetics. 1994 Feb;6(2):130-5.
This is the figure of STR loci of nuclear DNA from the Gill's review article in 2004. 
This is acknowledgement section of Gill's AA paper. It clearly states that hair sample was from Peter Kurth. 
Anastasia, Nyet.
Authors: Glausiusz, Josie
Source: Discover; Jan1995, Vol. 16 Issue 1, p99, 1/2p, 2bw
Document Type: Article
Subject Terms: ANDERSON, Anna
IMPOSTORS & imposture
ANASTASIA Nikolaevna, Grand Duchess, daughter of Nicholas II, Emperor of Russia
NICHOLAS II, Emperor of Russia, 1868-1918
Abstract:
Reveals that Anna Anderson, the woman who claimed to be Grand Duchess Anastasia, daughter of Russian Czar Nicholas II and Empress Alexandra who were executed in 1918, was a fraud. Medical evidence refuting the woman's claim; True identity of the impostor.
ISSN: 0274-7529
Persistent link to this record:
http://search.ebscohost.com/login.aspx?direct=true&db=mih&AN=9412224085&site=srck5-live
ANASTASIA, NYET
Section: GENETICS - 1994
IN OCTOBER RESEARCHERS at the British Forensic Science Service announced that by reading the entrails of a woman dead for the past ten years they had solved one of the century's most enduring mysteries. Not only, they said, was Anna Anderson not the Grand Duchess Anastasia--daughter of Czar Nicholas II and his wife, Alexandra, who were executed in 1918--she was likely to have been one Franziska Schanzkowska, a German-Polish factory worker who had disappeared in 1920.
It was also in 1920, in Berlin, that the woman called Anna Anderson first appeared, in a mental hospital; she'd been placed there after being dragged from a canal, an unsuccessful suicide. She made her claim to be Anastasia two years later, and until her death in the United States in 1984, she never wavered.
Not until this past year, though, could her claim be unequivocally repudiated. Peter Gill and his colleagues extracted DNA from a section of Anderson's intestine that had been preserved at a Charlottesville, Virginia, hospital after she had undergone surgery there in 1979. They found her chromosomal [nuclear] DNA to be lacking in pivotal sequences identified last year in DNA extracted from the bones of the czar and czarina.
The researchers also looked at Anderson's mitochondrial DNA--genetic material passed unchanged from mother to child--and compared its sequences with those of a maternally descended great-nephew of Schanzkowska's. They were identical.
By Josie Glausiusz
Legal proof of the validity of the DNA testing
....they (Anderson supporters) make stuff up to
support their case, without addressing the facts...
I for one am truly sick and tired of them saying
that this scientific analysis is "faulty" "unreliable"
and "inadmissable in courts" when it is WITHOUT DOUBT not the case!
In fact, one of the scientists who testified in this case, Dr. Terry
Melton, who determined by her mtdna research that AA was NOT GD
Anastasia is expressly found to be a reliable and credible witness
here, her work in the AA case is found to be STILL valid and reliable,
and this Court legally finds that her research is STILL reliable,
credible and admissable. I specifically wish to point out the
numerous times where each scientist testified UNDER OATH that they
were unaware of any scientific peer review studies which disagreed
with the accuracy and vailidity of their mtdna analyses and
methodology, as well as the numerous jurisditions which have found
this mtdna reseach admissible in evidence. This decision is from
September 2000 and has NOT been overturned:
PEOPLE v. KLINGER
713 N.Y.S.2d 823
N.Y.Co.Ct., 2000
Sept. 5, 2000
Judge Brown
PEOPLE v. MICHAEL KLINGER and RAYMOND KLINGER QDS:76703137—The
following constitutes the opinion, decision and order of the court.
***
By previous order of the Honorable Paul E. Kowtna, this court
conducted a Frye hearing on June 6, 2000 and June 13, 2000, to
determine the admissibility of mitochondrial DNA evidence at the trial
of the above-captioned Indictment.
At the hearing, the court heard testimony from two witnesses, Bruce
Budowle, Ph.D., a Senior Scientist with the Federal Bureau of
Investigation, and Terry Melton, PhD., President of Mitotyping
Technologies, LLC.
The court finds that Dr, Budowle and Dr. Melton were credible
witnesses.
The court makes the following conclusions of law:
The Court of Appeals has held that "[t]he long recognized rule of Frye
v. United States, 293 F. 1013, is that expert testimony based on
scientific principles or procedures is admissible but only after a
principle or procedure has 'gained general acceptance' in its
specified
field". In Frye (supra at 1014) the court stated:
"Just when a scientific principle or discovery crosses the line
between the
experimental and demonstrable stages is difficult to define. Somewhere
in
this twilight zone the evidential force of the principle must be
recognized,
and while courts will go a long way in admitting expert testimony
deduced
from a well-recognized scientific principle or discovery, the thing
from
which the deduction is made must be sufficiently established to have
gained general acceptance in the particular field in which it belongs"
(emphasis supplied)." (People v. Wesley, 83 NY2d 417).
"This Court has noted that the particular procedure need not be
'unanimously indorsed' by the scientific community but must be
'generally acceptable as reliable' (see People v. Middleton, 54 NY2d
42, 49). Thus the issue here concerns the acceptance by the relevant
scientific community of the reliability of DNA evidence." (People v.
Wesley, supra at 423).
"Once Frye has been satisfied, the question is 'whether the accepted
techniques were employed by the experts in this case" (People v.
Wesley, supra, citing People v. Middleton, 54 NY2d at 50). The focus
moves from the general reliability of the procedures followed to
generate the evidence proffered and whether they establish a
foundation for the reception of the evidence at trial. The trial court
determines, as a preliminary matter of law, whether an adequate
foundation for the admissibility of this particular evidence has been
established." (People v. Wesley, supra at 429).
The first witness was Dr. Bruce Budowle. Dr. Budowle has been employed
by the FBI for 17 years and has been a Senior Scientist for the past
one and a half to two years. He has a Ph.D. in genetics and a
Bachelor's Degree in biology, Dr. Budowle is a member of numerous
professional organizations including the American Academy of Forensic
Sciences and the International Society of Forensic Genetics. He has
published approximately 200-250 articles or materials relating to DNA
analysis, nine of those articles regarding mitochondrial DNA
(hereinafter "mtDNA"), The majority of these articles were subject to
peer review. Dr. Budowle has presented his research and findings to
the
International Symposium of Human Identification on nine separate
occasions. He explained that a symposium is a way to bring the
scientific community together so theycan exchange ideas. He also
serves on numerous journal and editorial boards both in this country
and abroad. Dr. Budowle has received numerous honors and awards
including the Forensic Scientist of the Year Award. He teaches a
course on mtDNA typing for the FBI and for Forensic Institute, which
is for national and international students. Dr. Budowle
has been qualified on numerous occasions as an expert witness in
molecular biology, genetics, population genetics, statistics and
forensic science in state, local and federal courts. He stated that he
has testified in more than half of the states in this country. Dr,
Budowle has also been qualified as an expert on mtDNA in New York,
Louisiana, Pennsylvania, Maryland and California.
As early as 1989, Dr. Budowle co-wrote a chapter of a book describing
mtDNA as a possible genetic tool. In October of 1993, he co-wrote one
of the first guidelines for the use of mtDNA sequencing in forensic
science. In 1995, he co-wrote a peer review journal
describing the procedure that was developed at the FBI for the
extraction, amplification and sequencing of mtDNA from human hair
shafts, Also, in 1995, a peer review article was co-written by him on
the validation of the aforesaid procedures for their application to
case work. An article was also co-written by Dr. Budowle, which was
published in 1997, that described a phenomenon observed in mtDNA
called heteroplasmy. Dr. Budowle also co-wrote a peer review article
for publication where a mtDNA study was done with crab
lice. He determined that this study was a valuable way of looking at
the DNA environment to determine whether its analysis produces a
reliable result. In 1999, he co-wrote a peer review journal article
describing some of the population data from a portion of the data
bases that demonstrates, by inference, the rarity of the mtDNA type
among unrelated individuals. Finally, Dr. Budowle is on the DNA
Commission of the International Society for Forensic Genetics. He was
one of 13 members of the DNA Commission who published an
editorial which contained guidelines for typing mtDNA.
***
MtDNA is much heartier than nuclear DNA. For example, old bones and
teeth that have been exposed to the environment may still have
sufficient quantity for mtDNA typing where nuclear DNA typing would
fail to give a result, There are, however, differences between the two
types of DNA. First, in nuclear DNA, you inherit half from your mother
and half from your father. In mtDNA, you inherit all of it from
your mother. Second, instead of being billions of letters long, the
mtDNA strand is 16,569 letters long. Further, mtDNA is circular rather
than linear. Dr. Budowle opined that the circular strands may actually
protect the mtDNA from being degraded.
***
the counting method is used to predict how common a particular
profile is in mtDNA. Next, the technician can go further by
calculating a confidence level based upon a statistical formula
established early in the twentieth century. The lab, in essence, would
calculate a confidence interval around the estimated frequency based
on the size of the database. This formula is based upon bell-shaped
distribution theories that have been in existence since the
mid-eighteenth century. A confidence level, based upon a statistical
analysis, creates an upper bound to the benefit of the accused, and
then provides that they have confidence that the frequency is no
higher than this amount, Dr. Budowle is not aware of any peer review article
that disagrees with this method of calculation.
MtDNA research began at the FBI in 1992 and testing commenced in 1996.
Numerous procedures and protocols were developed that were subject to
peer review. Moreover, validation studies for mtDNA have been
published and subject to peer review.
Apparently, there have been no peer review articles that disagree with
the FBI validation ...
DNA and the law: Anderson supporters are very wrong in their assertations that the mtDNA testing would not hold up in court!
List of court cases where mtDNA testing was upheld
http://www.denverda.org/DNA/Mitochondrial_DNA_Legal_Decisions.htm
So much for the shoes and ears!
Proof that DNA overrules all other evidence in a court of law
http://innocenceproject.org
***********************************
More on the DNA testing done and its realibility:
Dr. Terry Melton
Dr. Terry Melton has been working with mtDNA since 1991. She has a
Ph.D. from Penn State University in genetics. She has performed
hundreds of DNA analyses and thousands of PCR amplifications. She
testified that her lab exclusively performs mtDNA analysis. One
high profile analysis that she was involved with was the claim of Anna
Anderson that she was the remaining living child of the Romanov
family. By the use of mtDNA, it was determined that she was not the
Grand Duchess Anastasia.
The Court cited the AA case here as support for Dr. Melton's
credentials as an expert in mtDNA analysis. Please note that there is
no reference to any question of the accuracy of the results of the
test. In fact, the accuracy of the AA test is overtly IMPLIED because
the Court cites it as part of the basis for believing her expertise in
mtDNA work and IN FACT RELIES ON THIS CASE to demonstrate her
expertise.
In plain English, what this says is that NO SCIENTISTS have published
ANYTHING to date which has questioned the accuracy of her work,
INCLUDING the mtDNA analysis of AA, excluding her as GD Anastasia. I
have seen lots of claims made here of the "innacuracy" of the AA mtDNA
testing, but for some reason, NO ONE can actually
cite any published scientific reference to back up their claims.
Dr. Melton testified that, in her opinion, the underlying principles
of mtDNA, the principles of mtDNA analysis and the statistical methods
as applied to mtDNA are generally accepted as reliable in the
scientific community.
In plain English again, mtDNA analysis, IS regarded as reliable and
accepted by the scientific community.
Here it is, folks, accepted as a matter of law by
the Supreme Court of New York:
The court finds that the credible evidence adduced at the hearing
established that mtDTA analysis and interpretations are generally
accepted as reliable in the scientific community and that the
procedures followed in this case establish a foundation for admission
of such evidence. The evidence has sufficiently established that the
analyses and interpretations of mtDNA has gained general acceptance in
the community of scientists that work in this field. The existence of
contamination and heteroplasmy do not affect the reliability of the
scientific procedure and these issues, which are subj ect to
cross-examination at the time of trial, do not invalidate the
procedures of mtDNA testing.
Although both Dr. Budowle and Dr. Melton testified that mtDNA can not
be the unique identifier that nuclear DNA can achieve, this conclusion,
however, does not invalidate the accuracy of the procedure and whether
it is acceptable in the relevant scientific community.
This court finds that many of the procedures used in analyzing mtDNA
are the same as those used in analyzing nuclear DNA. Further, the
statistical methods used by the technician in creating the upper
bounds of the confidence interval are basic statistical methods that
have been found generally accepted in the relevant scientific
community.
Moreover, mtDNA procedures have been subject to peer review and Dr.
Budowle testified that he knew of no peer review articles that state
that the aforesaid process and statistical methods were not
scientifically reliable. In addition, Dr. Melton testified that the
whole process has been subject to peer review and that she is unaware
of any peer review articles in disagreement with the methods used by
her lab with respect to analysis, interpretation and use of the
statistical formulas.
Once again, Dr. Melton said that NO SCIENTIST has ever questioned in
writing the accuracy of her results or methods in any of her work,
which expressly includes her work EXCLUDING AA as Anastasia. So if the
AA mtDNA analysis is so flawed and unreliable, WHY havent any
scientists stated so in writing??
"She (Dr. Melton) testified that her lab exclusively performs mtDNA
analysis. One high profile analysis that she was involved with was the
claim of Anna Anderson that she was the remaining living child of the
Romanov family. By the use of mtDNA, it was determined that she was
NOT (my emphasis here) the Grand Duchess Anastasia. ...
Dr. Melton testified that she is unaware of any peer review articles
in disagreement with the method used by her lab with respect to the
analysis and interpretation of mtDNA. She testified that there is no
process for mtDNA analysis that is not generally accepted as a valid
scientific procedure. The whole process has been subject to peer
review. Further, the statistical formula for mtDNA is generally
accepted by the scientific community. Dr. Melton testified that there
were no peer review articles stating that this statistical formula or
method was not a reliable interpretation of the mtDNA database. She
also testified that the counting method, the confidence interval
approach and the likelihood calculation are each equally valid."
While mtDNA analysis can not necessarily prove who a single individual
may be, (it cannot go beyond 99.9%, which is the probability AA is FS)
it CAN prove who they can NOT be......reliably so by
excluding them from the possibility of blood relation in the maternal
line. There is NO QUESTION that Anna Anderson was NOT Grand
Duchess Anastasia Nicholaiovna because her mtDNA was EXCLUDED from
relationship thru the maternal line. The fundemantal issue here
remains that mtDNA analysis has proved reliably that AA could not have
been GD Anastasia.
***********************************
Explaination of the science of the DNA testing from someone who met Dr. Melton:
I have spoken to Dr. Teri Melton of Mitotyping Technologies, who did the original Anna Manahan/Carl Maucher testing. There is no doubt, in her mind, or anyone of the other scientists that the original sample could NOT have been "corrupted" and as far as they are all concerned, in her exact words "there is no need to re test the samples as you will get the exact same results".
The reason the samples could not have been corrupted is simple. The Anna Manahan sample was tested first and sequenced BEFORE the Carl Maucher sample was even taken or sequenced. As a result, nobody could have possible known the Maucher sequence OR contaminated the Anna Manahan sample with Maucher mtDNA. The fact that the Anna Manahan sample was a 99.5% likelihood MATCH to the Carl Maucher DNA is of itself proof to the scientific community that the testing was accurate. You see, simply put, if the sample was "corrupted" there couldn't have been any sequence stable enough to sequence; "if" the sample was "tainted" by outside DNA it would have never matched the Maucher sample to such a high degree of certainty (unless one of the scientists handling the sample was themselves a very close cousin to Maucher (they weren't) and the fact that FOUR different labs all got the exact same results rules out the possibility of corruption, contamination or scientific error.
The science is simple and clear. Anyone who thinks the Anna Manahan testing was corrupted or contaminated simply just doesn't grasp the simple science of it all.
My own personal response from Dr. Melton herself!!(quoted with her permission)
As in all fascinating historical mysteries, conspiracy theories will abound. I can address only the lab process.
My response to you is the same that I give to everyone who questions the legitimacy of the Anderson results:
Multiple labs got the same results on different tissues (hair/intestinal tissues) at different times. Independent testing such as this is best practice in forensic testing, especially when the results are going to be scrutinized at the level of this case. It is highly unlikely that the same results would be obtained in different labs if the work was shoddy. More likely, the labs would have gotten different results that made no sense compared to each other.
The science that was used is basic, and the methods, while becoming more sensitive and streamlined since the time of the original tests, were and are designed to get at the most basic building blocks of human identity: the DNA sequence. The DNA sequence cannot change when the methods change. There is no more elemental level of inspection.
Conspiracy theories don't worry me. The weight of well-conceived and time-tested protocols carried out by laboratories with impeccable credentials and nothing to gain from either answer are behind all the results, which have been published in scientific, peer-reviewed literature.
I hope this helps.
Best wishes,
Terry Melton
Mitotyping Technologies information and website
Dr. Terry Melton
http://www.mitotyping.com/598591928115356/blank/browse.asp?a=383&BMDRN=2000&BCOB=0&c=51341&598591928115356Nav=%7C26%7C&NodeID=26
Security and chain of custody explainations
http://www.mitotyping.com/598591928115356/blank/browse.asp?a=383&BMDRN=2000&BCOB=0&c=51409&598591928115356Nav=|&NodeID=72
.
|
***********************************
Excellent article on the bones found in 1991, now buried in the fortress:
http://www.geocities.com/mushkah/Nagai.html
************************************
So, there you go. Despite all their bold assertations, no one, especially not any Anna Anderson supporters, have been able to prove the DNA tests invalid, inaccurate, or that the sample was not that of Mrs. Manahan aka "Anna Anderson." Of course they're never going to stop saying the DNA is flawed, but proving it against courts and scientific peer review is quite a different matter. So don't believe their hype on this either. Science and history have spoken.
National Geographic Explorer "Finding Anastasia" explains DNA results
Solving One of The Great Mysteries Of The 20th Century: National Geographic
Channel's Explorer: Finding Anastasia Provides Exclusive Coverage of The
Forensic Investigation of Romanov Family Remains
90 Years After the Murders: Forensic Experts Conclusively Prove That
Tsar's Missing Children Are All Accounted For
Explorer: Finding Anastasia premieres Tuesday, July 22, 2008, at 10 PM ET/PT
WASHINGTON, July 10 /PRNewswire-USNewswire/ -- On July 17, 1918, Bolshevik
revolutionaries gunned down Russia's Tsar Nicholas II and his family in a
Siberian home. But the events of that night have remained shrouded in mystery
and controversy. Rumors persisted that some in the family might have escaped,
including the tsar's youngest daughter, Grand Duchess Anastasia, her sister
Grand Duchess Maria and their brother Crown Prince Alexei, the 13-year-old
heir to the throne. In 1991, the unearthing of remains in a remote forest
grave suggested that the Romanovs were found -- most of them, at least. The
bones of Alexei and one of the sisters were missing. Now, a recently
uncovered second grave reveals the truth behind the royal family's final
grisly chapter: None of them got out alive.
In conjunction with the 90th anniversary of the assassination, National
Geographic Channel's (NGC) Explorer goes inside the investigation with
exclusive access to the forensic investigation. Premiering Tuesday, July 22,
2008, at 10 PM ET/PT, Explorer: Finding Anastasia puts the mystery of the
Romanov family's fate to rest. Working in collaboration with the Russian
government, American investigator Dr.Anthony Falsetti, a veteran of the 9/11
investigation, travels to Russia to examine the remains. Garnering DNA,
ballistics and the very latest forensic analysis, an international team of
scientists, including Dr. Michael Coble, one of the world's leading forensic
DNA experts, labors to find the truth.
In 1991, a media firestorm surrounded the discovery of the first set of
Romanov bones and subsequent controversial findings. In the end, the Russian
Orthodox Church rejected DNA evidence as tainted and refused to acknowledge
that the remains were in fact the Romanovs. Dr. Falsetti was part of the
original investigative team, and now, he hopes the second gravesite discovery,
found just 70 yards from the first, will end all speculation on the Romanovs'
fate.
The newly uncovered bones undergo intense 21st century forensic science
scrutiny, but not without plenty of obstacles. First, Dr. Falsetti and Dr.
Coble discover that the 44 bone fragments are severely broken, almost
unrecognizable, and that they show evidence of burning. Confirming the unique
royal genes in the fragments will be painstaking and require that the remains
be carefully sent from Russia to the United States.
Separate from the DNA analysis, Dr. Falsetti reviews artifacts such as bullets
recovered from the gravesite and combs through the archival accounts of the
executioners to see if the history and bones tell a common story. The newly
discovered bullets turn out to be nearly identical to those found in the first
grave.
Then, Explorer: Finding Anastasia unfolds the gruesome story of why two bodies
would be separated from the others, and in the process, finds that some of the
greatest savagery by the Bolsheviks likely began when the shooting finished.
Experts now believe that first, the bodies were thrown down a mine shaft --
only to be retrieved shortly after the burial location was leaked to the
locals. The perpetrators then planned to switch locations and incinerate two
of the corpses. Because of the lengthy burning process, they dismembered the
corpses and covered them with acid to disfigure them beyond recognition.
Running out of time before dawn, they threw the other nine bodies into another
hole and covered them in acid.
Cuts and burn marks in the newly discovered bones corroborate a story of the
executioners' barbaric acts. Falsetti is haunted by the story. "I am a
scientist but I am also a father. And this is a horrible, terrible thing to
have happened to very young people," he says.
Five months after first selecting the bone samples for analysis, Dr. Coble is
now ready to share his findings on DNA. The evidence is overwhelming that the
bone fragments belong to Crown Prince Alexei and one of his sisters. That
sister was either Maria or Anastasia. But beyond that the DNA is silent. One
thing is for sure: The entire Romanov family is accounted for. And despite
the persistent claims, Anastasia, the youngest Romanov daughter, did not
escape.
SOURCE National Geographic Channel
http://www.reuters.com/article/pressRelease/idUS160385+10-Jul-2008+PRN20080710
Explorer: Finding Anastasia is produced for the National Geographic Channel by
National Geographic Television (NGT). For NGT senior producer is Robert
Zakin, and executive producer is Jonathan Halperin. For NGC executive
producer is Kathleen Cromley, senior vice president of production and
development is Juliet Blake, and executive vice president of content is Steve
Burns.
Based at the National Geographic Society headquarters in Washington, D.C., the
National Geographic Channel (NGC) is a joint venture between National
Geographic Ventures (NGV) and Fox Cable Networks (FCN). Since launching in
January 2001, NGC initially earned some of the fastest distribution growth in
the history of cable and more recently the fastest ratings growth in
television. The network celebrated its fifth anniversary January 2006 with
the launch of NGC HD which provides the spectacular imagery that National
Geographic is known for in stunning high-definition. NGC has carriage with
all of the nation's major cable and satellite television providers, making it
currently available to nearly 68 million homes. For more information, please
visit www.natgeotv.com.
SOURCE National Geographic Channel
Russell Howard, +1-202-912-6652, RHoward@natgeochannel.com, or Chris Albert,
+1-202-912-6526, CAlbert@natgeochannel.com, or National Broadcast: Dara Klatt,
+1-202-912-6720, Dara.Klatt@natgeochannel.com, all of National Geographic
Channel; or National & Local Radio: Johanna Ramos Boyer, 703-646-5137,
Johanna@jrbcomm.com, or National Print: Christie Parell, +1-202-496-2124,
CParell@fratelli.com, or Local Print: Licet Ariza, +1-202-496-2122,
LAriza@fratelli.com, for National Geographic Channel
Article explaining the DNA testing-proof they have the entire family now
By Fred W. Baker III
American Forces Press Service
WASHINGTON, July 15, 2008 – A Defense Department DNA identification lab has helped bring to a close a near-century-old mystery, laying to rest a search for the remains of two children executed alongside the rest of the family of Russia’s last czar.
In the midst of the Bolshevik Revolution in 1918, the imprisoned Nicholas II, his wife and five children were shot and killed along with four loyal servants in the basement of a merchant’s house. The ruling Bolshevik party eventually would become the communist party of the Soviet Union.
What happened to the bodies remained somewhat of a mystery for years. Rumors circulated of survivors of the execution. Hundreds came forward over time claiming to be a surviving member of the royal family. The most prominent was Anna Anderson in 1920, who claimed to be the czar's youngest daughter, Anastasia.
More than a decade ago, the Russian government asked the Armed Forces DNA Identification Lab in Rockville, Md., to use its cutting-edge technology in DNA testing to help confirm the identify the royal family’s remains after a mass grave was discovered. The lab positively identified the remains of the czar, his wife and three daughters, and later disproved Anderson’s claims of royal heritage.
That “put the lab on the map” in terms of leading the field in such testing, said Army Lt. Col. Louis N. Finelli, chief deputy medical examiner and director of the DoD DNA registry.
“That’s our index case. It launched us from a fledgling mitochondrial DNA laboratory to one of the premier laboratories,” Finelli said.
But the remains of two children, a boy and a girl, were still missing, opening the door for some to speculate that maybe there were survivors of the execution.
Now, the lab has again helped the Russian government by identifying the remains of those two children, found last year in a shallow grave about 70 feet from the larger gravesite.
“There’s no doubt that nobody escaped,” said Dr. Michael Coble, the research section chief for the lab. “We can conclude here that based on this evidence, we have [recovered] the two missing children.”
Originally, according to historical accounts, the bodies of the family were thrown down a mine shaft in the town of Yekaterinburg, where they where killed. But Bolshevik leaders feared that a counterrevolutionary movement would recover the bodies.
According to accounts, a truck that was moving the remains to another location broke down en route, about 12 miles north of Yekaterinburg. It was decided to destroy the remains there.
Wanting to ensure the bodies were not recovered, the Bolsheviks first tried to destroy the remains of two of the children. They chopped up the bodies, set them on fire, and poured sulfuric acid on them.
“They tried everything they could to completely dissolve the remains, and it didn’t work,” Coble said.
Because the exhaustive efforts did not yield the expected results, it was decided to bury the remaining bodies in another mass grave nearby. The bodies were doused with sulfuric acid and covered with dirt.
The larger grave was discovered in the 1970s, Coble said, but the remains weren’t recovered until 1991, after communism fell in the country.
The smaller grave was discovered last summer. At the site were telltale remains of bones, bullets and ceramic pieces of the containers that held sulfuric acid. Time and torture had taken their toll on the remains, though. Only 44 bone fragments remained in the grave, most so small they weren’t useful for DNA testing.
The Russian government contacted the DoD lab in October and asked them to help with the DNA identification. The lab is one of the oldest and largest labs working with “ancient” DNA testing, or testing from severely degraded samples, Coble said. The lab is used routinely to identify the remains of missing U.S. servicemembers not identifiable by means other than DNA testing.
Most commercial labs test DNA collected from hair samples, but the DoD lab works with more bone samples than any other lab in the country -- as many as 800 samples a year.
“We have one of the world’s greatest labs as far as doing these types of investigations,” Coble said.
The lab worked in conjunction with the Institute for Forensic Medicine in Innsbruck, Austria, and with a Russian scientist doing collaborative work at a lab at the University of Massachusetts.
Multiple labs testing the same remains and drawing the same conclusions ensures that the results are reliable and none of the evidence was contaminated, Coble said.
A handful of scientists and technicians at the DoD lab spent about a month’s worth of man-hours on the project. Tests from fresh DNA samples can be turned around in a day, but with these cases, the analysis can take much longer.
The lab received 10 samples, most in poor condition. Some were thin skull fragments. By the time they were sanded, scrubbed and cleaned up, there was little left for testing; some were only the size of a dime.
Two main fragments, though, were about a half inch thick and a couple of inches in diameter. They were thigh bones, hollow in the middle, one from a male and one from a female. Leg bone samples are best for DNA testing, Coble said, because they are thick and dense.
Mark Wadhams, assistant technical leader for the mitochondrial DNA section, worked on the case. He said that once the bones are cleaned, they are ground into a fine powder, about the consistency of fine sand, and an “extraction” buffer is added. The extraction buffer dissolves the bone powder into liquid and allows the release of the biological material.
Scientists then remove the DNA from the liquid, to get the DNA extract. The sample is reduced from about 3 milliliters of volume to about 200 microliters of clear liquid in a tube.
The biggest potential contaminate to the samples is the lab workers themselves.
“Since your body sloughs off probably over 400,000 cells per day, you have a potential to get into that sample pretty easily,” Wadhams said.
The amount of DNA collected depends on the quality of the sample. From the nearly 100-year-old samples, not a lot was expected, Wadhams said.
“The samples were much smaller and much more degraded. You never know by looking at a sample whether it’s going to work or not, so you basically have to go through the entire process to see if it will work,” Wadhams said.
The DoD lab scientists tested the remains three ways.
First they tested its mitochondrial DNA, which is passed from the mother to her children. Using this testing, they established that both remains had the same mitochondrial sequence as that recovered earlier from the czar’s wife’s remains.
The lab scientists now knew, based on the initial DNA testing on the remains of the mother and children in the larger grave, that they had strong evidence that these were the remains of the two missing siblings.
But mitochondrial DNA is not unique to an individual, in that the testing proves only relationship, not necessarily identity.
So the lab then looked at the nuclear DNA of the samples. Everyone gets half of their nuclear DNA from each parent. This data showed that the samples were from a male and female who shared some DNA.
In fact, the data showed that it was more than 5 million times more likely they were related to each other than not, Coble said. Scientists are typically convinced of relationship with a likelihood ratio greater than 500, he said.
Then, to prove that the remains were the missing siblings of the Russian royal family, the lab went back to samples from the larger gravesite and developed DNA profiles of the other siblings and parents.
When the samples were matched, the data showed that the likelihood they were members of the royal family was more than 4 trillion times the likelihood they were not. A typically strong likelihood ratio would be more than 1,000, Coble said.
Finally, the lab tested the Y chromosome of the male sample. That test again confirmed that the remains of the male child indeed were those of the czar’s only son.
“We have very strong evidence on all three marker systems that what have here are the [remains of the] missing children,” Coble said.
The lab delivered a preliminary report to the Russian government about a month ago, detailing the findings of the lab. The results were officially released today.
Finelli said this type of cooperation between the two governments “transcends the politics of what may be the relationship between the two countries.” And, he added, the lessons learned from these types of cases are applied to the daily mission of the lab.
“These types of cases are so challenging that we can apply what we learn in those cases to the current-day effort of [the servicemember identification] mission,” Finelli said. “We test ourselves with these very hard cases, and then we can apply it to the casework to account for our fallen. So it’s always a little bit of a selfish motivation"
http://www.defenselink.mil/news/newsarticle.aspx?id=50507
Putting the last questions to rest
http://www.defenselink.mil/news/newsarticle.aspx?id=50507
This article looks like it answers all the questions.
DefenseLink News Article: DoD Lab Helps to Resolve Century-Old Russian Mystery
Questions answered by this article:
1. Is there a definitive declaration that everyone died?
Answer: YES
Quote from article:
“There’s no doubt that nobody escaped,” said Dr. Michael Coble, the research section chief for the lab. “We can conclude here that based on this evidence, we have [recovered] the two missing children.”
2. What bones were used for testing? (it's been suggested maybe it was only a tooth, or perhaps something from the other grave)
Answer: Leg bones of the male and female. Not a tooth for Alexei, and since no leg bones are missing from the first three girls, this one is definitely the fourth and missing girl.(see photo of the boy's leg bone in the article)
Quote from article:
Two main fragments, though, were about a half inch thick and a couple of inches in diameter. They were thigh bones, hollow in the middle, one from a male and one from a female. Leg bone samples are best for DNA testing, Coble said, because they are thick and dense.
3. Were separate profiles made for each girl?
Answer: YES, one was made for each member of the family. They already had profiles for the two 2007 bodies, and then made profiles of the parents and other three girls.
Quote from article:
Then, to prove that the remains were the missing siblings of the Russian royal family, the lab went back to samples from the larger gravesite and developed DNA profiles of the other siblings and parents.
4. Does this mean we can tell which girl was missing and which was in the grave, Anastasia or Maria?
Answer: No, because there is no nuclear DNA sample preserved from either of those girls when they were alive to compare to the nuclear DNA found in the bones. However, there were different profiles for all the children and all four girls were proven to have died that night.
5. What are the odds there was some mistake, and these are only random people murdered and dumped in same area the with similar DNA?
Quotes from article:
So the lab then looked at the nuclear DNA of the samples. Everyone gets half of their nuclear DNA from each parent. This data showed that the samples were from a male and female who shared some DNA.
In fact, the data showed that it was more than 5 million times more likely they were related to each other than not, Coble said. Scientists are typically convinced of relationship with a likelihood ratio greater than 500, he said.
The chances of the two bodies in the burn pit NOT being siblings is 5 million to one. That is far more than the 500 to one that qualifies as convincing to scientists.
6.What are the odds these are just two siblings who happened to be dumped there and are not related to the royal family?
Quote from the article:
When the samples were matched, the data showed that the likelihood they were members of the royal family was more than 4 trillion times the likelihood they were not. A typically strong likelihood ratio would be more than 1,000, Coble said.
The odds they were not related to the family in the mass grave are FOUR TRILLION TO ONE- and it only takes 1000 to 1 to convince scientists. So as you can see the odds are astronomically in favor of these being our Romanovs, and all the scientists are completely convinced they have found all the children.
7. What type of DNA testing was done?
Answer: (from info in the article) ALL 3 types- mtDNA and nuclear on the male and the female, and Y chromosome for the male. You will see the details spelled out in the article. All forms of testing gave the same conclusions, very strong evidence that the bones were from the 2 missing Romanov children.
8. What are the names of the labs involved?
According to the article:
US Department of Defense DNA Lab
Institute for Forensic Medicine in Innsbruck, Austria,
a Russian scientist doing collaborative work at a lab at the University of Massachusetts.
9. Some claimant supporters have asked: "How do we know this isn't just more of the Russians putting a spin on things?"
Answer: This information in this article came from an American scientist who works for the Dept of Defense lab, and his work has matched the ones done in Austria and by the Russian working in Mass. So there's no way to blame the Russian officials for anything here. Yes, perhaps the Russian officials were not as thorough as they should have been in their latest announcements, maybe they felt they didn't need to be. But getting the real deal straight from the horse's mouth of the guy who did the tests tells the story. The Russians, or anyone else, have no reason to lie or hide anything. Some people may not like the results but they are accurate. Though conpiracy theories will likely continue among a few diehard claimant supporters, they will be no more than fantasies of no value.
10. Does this mean an end to all claimant possibilities?
Answer: YES
These details explained in the article put the last nail in the coffin of hope for claimants. Now since all the bodies have been found and there's proof no one escaped, it doesn't even matter if AA's intestines were switched or not!
So as you can see there is no doubt about the authenticity of the bones and fragments tested. They are the Romanovs, all 7 of them.
No matter what outcome we had hoped for, now we can all put this issue behind us and move on and accept these test results and the answers they provide. Instead of trying to find reasons not to believe them, we should honor and respect the scientists and labs who worked so hard to get these results for us, and thank them for their historic conclusions.
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