Conversations With the Experts: DNA Forensic Testing in New York City

Peter Jhon

Disclosures

October 30, 2001

Introduction

Medscape recently spoke with Brian Ward, PhD, Vice President of Operations for Myriad Genetic Laboratories, Inc., in Salt Lake City, Utah, about the difficult task of identifying the victims of the World Trade Center disaster. Myriad Genetics is 1 of 3 companies performing DNA analysis of the victims. All 3 companies are working in conjunction with the New York City Medical Examiner's Office.

Q: What is Myriad Genetics' role in the DNA analysis of the victims of the World Trade Center attacks?

A: The New York State Police Forensic Laboratory is receiving cheek swabs from the relatives of the victims, as well as personal effects of the victims. At the site of the World Trade Center, the New York City Medical Examiner is receiving the victims' specimens. These 2 groups catalogue and run standard forensic tests on the specimens. Then the DNA from the samples is extracted and sent to Myriad. We analyze the samples and create profiles known as DNA fingerprints. This information is sent back to New York, where the kinship analysis takes place -- when the victim is matched with family members.

Q: What other companies are involved with this project?

A: Three biotechnology/biopharmaceutical companies are involved: Celera (Rockville, Maryland), who will perform mitochondrial DNA (mtDNA) analysis; Bode Technology (Springfield, Virginia), who will be assisting in the extraction of bone as well as quality assurance; and Myriad Genetics, who will perform short tandem repeat (STR) analysis.

STR analysis is very accurate, and the matches are made at 1 in a million, or at higher stringency. Mitochondrial DNA is a different type, and the matches are not as stringent. Our preference is to do STR samples.

Q: What is the difference between STR technology and mtDNA tests?

A: For STR analysis there are 14 recognized markers -- 13 markers plus the sex chromosome. The chance that you and I have the same markers is 1 in 250 trillion, so when reassociation of dismembered body parts is accomplished, it is with an extremely high level of confidence. When kinship analysis is performed to match the victim with the family, the 14 victim markers are matched against DNA of the mother and father. Our DNA is a mixture of the DNA from our mom and dad; by doing comparisons backwards, we can take a victim's STR profile and match it to the parents'. The accuracy of that is at least 1 in 3000, to well above 1 in a million to even 1 in a billion, so this is highly accurate.

Mitochondrial DNA is only inherited from the mother, and the DNA itself is only about 80% informative. So it is generally used as a back-up, when STR profiles do not work. The advantage of mitochondrial DNA is that it can be extracted from bone, and can be amplified from very small amounts of samples.

In biological tissue, you have 2 copies of each STR marker, but you have thousands of mitochondrial cells. But unfortunately the mitochondria are very similar, and the information content is much less than that of a STR profile.

Q: Could you describe the process of analysis?

A: Let me give you an example. To do a breast cancer analysis, we have to study 37,000 base pairs per patient and proofread every single base pair. So we established a system that is driven with a centralized database using robots and high-sequencing throughput machines, connected to proprietary software for our analysis.

We get a bit of DNA in the laboratory, and establish an electronic chain of custody so that every step/reagent/plate/technologist that interacts with that sample is recorded. We amplify specific regions of that DNA using polymerase chain reaction (PCR) amplification, and amplify 1000 copies of a particular region of a chromosome to millions of copies. We then separate out specific regions of chromosomes by size, and those are fluorescently tagged so we can look at specific sizes of regions. In those regions we look for repeating elements -- 4 base pairs repeating over and over. So this is not gene DNA, this is garbage or spacer DNA. For example, my spacer DNA on chromosome 13 may have a repeating pattern of 12 on one chromosome and 15 on another, and yours might have one of 7 and 5. We create a numeric printout of all of the spacing size of these regions, which is analyzed by our technologist. That number profile is then sent to New York for identification.

Representative short tandem repeat (STR) gel image. After the DNA is isolated and amplified in the presence of specific fluorescent tags, the different sizes of DNA from all of the markers are separated on electrophoresis gel. This is a snapshot of that separation gel. It has 88 specimens (plus controls); each vertical lane comprises 10 different markers from 1 specimen. Published with permission of Brian Ward, PhD, Myriad Genetics, Inc.; Salt Lake City, Utah.

Representative allele calls. Fluorescent markers from the gel are transformed into a wave form and presented on a computer screen, so that a data analyst can examine and make the specific allele calls that comprise the STR profile. Published with permission of Brian Ward, PhD, Myriad Genetics, Inc.; Salt Lake City, Utah.

Sample allele calls. This is the summary of all markers from 1 sample. There are a total of 14 markers per sample, some overlapping. The letters across the top are the specific markers: D13 is a marker on the 13th chromosome; D21 is from chromosome 21; Amelogenin is the sex chromosome pattern; D21 allele calls are 27.0 and 30.0; the CSF pattern is 11.0/11.0; and the FGA pattern is 18.0/20. Published with permission of Brian Ward, PhD, Myriad Genetics, Inc.; Salt Lake City, Utah.

Q: What are the specific difficulties of DNA analysis for this particular project?

A: The greatest difficulty is the bacterial breakdown of DNA and the putrefaction of skin. Generally, degraded DNA works very well for STR; you might not get all 14 markers, but if you have 9 or 10 markers, that is more than adequate for analysis. The largest challenge is actually finding a biological specimen that is persevered well enough to extract DNA (that is the greatest difficulty). Our experience has been that if there is DNA present, we are extremely successful in obtaining a profile.

If a victim's flesh is charred, or seared, so to speak, forensic pathologists can actually cut into the tissue, and it actually works as a preservative by searing and sterilizing the outside of the tissue. If the bodies are incinerated, like in a crematorium, the only thing that will remain will be bone. The chances of getting DNA from bone are small, but the chances of getting mitochondrial DNA are pretty good.

Q: What is the minimum sample size of DNA needed to run an analysis?

A: In the nanograms -- very small amounts are needed. In fact, our reaction mixtures are normalized at 3 micrograms per microliter.

Q: How long does it take to run 1 sample in the lab?

A: In a casework environment, you can run 1 sample in about 1.5 or 2 days; however, that is not what our environment is good at doing. If you brought a single sample into our lab and asked us to analyze it, that would be difficult. However, if you brought 1000 samples to us and asked us to analyze those samples in 2 weeks, our production environment works great. So in general, it takes approximately 2 weeks for us to cycle through all of the tests. We analyze each sample 4 times. We have 2 amplification programs, PROFILER and COFILER; we run both twice, then we run the proprietary software, SURELOCK -- that is the database and chain-of-command software used to analyze the data.

Q: What percentage of the victims will be identifiable?

A: I'm very hopeful that if biological samples are obtained, about 70% of them will be identifiable. However, unlike a plane crash, we don't have a manifest, we are not quite sure who is where, and it's difficult to get a relative's DNA. It's just unknown at this time. Unlike a plane crash, like at the Pentagon, they know that they have 283 or so victims, they can measure against that number, and they have a pretty good idea of who was there.

Q: How many samples does Myriad/New York City expect to run?

A: No one is really talking about the number of samples right now. The samples started to come in at the rate of approximately 1000 a day, then there was a short lull, and now things are back to about 1000 a day. Estimates range anywhere from 50,000 to 1.5 million samples.

Q: How long will this take?

A: The most telling statistic is the fact that 4 weeks after the attack, only 20% to 25% of the rubble pile has been moved. We are looking at at least another 4-6 months for recovery efforts, and another several months for DNA analysis. This is an ongoing project, and as the recovery gets longer and longer, the DNA is going to be much more difficult to analyze, because it will be degraded.

Q: Is there a cutoff time as to how long a sample will be viable?

A: No, it depends very much on the local conditions. But for the Swiss Air crash, where victims were immersed in 4° seawater for a month or so, [researchers] were very successful in extracting DNA. On the other hand, this is a very different type of crime scene because there is jet fuel, fires that are still burning, water, and rubble -- so nobody knows. The medical examiner's obligation is to run DNA analysis on any sample that is recovered.

Q: How much will this cost?

A: I'm glad you asked that question. I can truthfully say that we have had no meaningful conversations about the costs of these tests with anyone. Of course we know the actual costs of the tests, but that is proprietary information. Generally speaking, these sample types cost in the vicinity of $50 a sample. The collection, cataloging, and extraction of DNA can be 1-10 times more expensive than the tests that we run. Our challenge is the fact that we have had to create multiple new informatics pathways in our automated system in order to accept these samples. Our commitment is that we will not benefit financially from this endeavor. We think that it is unethical to make a profit on this work, although we are a for-profit company. We will be doing this at a modest, reasonable expense.

Q: Who is handling the DNA analysis in Washington, DC?

A: The Armed Forces DNA laboratories in Maryland.

Q: Is there any precedent of DNA forensic analysis of this scale?

A: This is anywhere from 20-40 times larger than anything that has ever been encountered. The Swiss Air crash was the first time this type of DNA analysis had been used in a forensics environment, and then there was also [the] Egypt Air [crash].

Q: Are there any concerns of "cross-contamination" between samples?

A: Yes, [because the samples are not extracted] in the laboratory. Our lab was designed for high-throughput genetic sequencing of breast, ovarian, and colon cancer patients, so we have built our robotic systems to manage that contamination. However, at the site, you can mix samples. So far we've seen very little contamination. That is in regard to STR contamination. However, the challenge is with mtDNA -- it requires much more amplification, so minute traces of contamination are really a problem.

Q: What advances in DNA analysis have been made in the past 5 years?

A: The technology has not actually changed that much, but about 4-5 years ago, the Technical Working Group on DNA for Forensics standardized the markers used for amplification in North America.

Q: What is the accuracy of the tests?

A: Once you get a match, the accuracy will be 999,999 out of a million, or higher. Sometimes you can get a match to identity that the person's profile belongs to a specific person, without a shadow of a doubt -- 100%.

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