Vietnam Veterans of America
Each of us has about thirty-seven trillion cells. “Even if you could count ten cells each second, it would take you tens of thousands of years to finish counting,” says Carl Zimmer, a leading science writer. “Perhaps most importantly, the very fact that some thirty-seven trillion cells can cooperate for decades, giving rise to a single human body instead of a chaotic war of selfish microbes, is amazing.”
The DNA, deoxyribonucleic acid, researchers rely on to identify the remains of those lost during wars and other calamities, is made of infinitesimally small molecules within those trillions of cells. Under an electron microscope some cells are easy to see and others are tangled so tightly they are difficult to identify. Cells range in size from 0.1 to 5.0 micrometers in diameter. An egg cell is big enough to be seen with the naked eye, far larger than a sperm cell, one of the smallest human cells. Inside all these cells are thousands of “machines,” as Zimmer calls them. In that miniscule space are the mechanisms that make you who you are.
Cells come in all shapes—rods, oblongs, squiggles, tadpoles, squares, stars, splats, dendrites, and other indescribable, complex forms with tails, arms, and other odd appendages.
There are multitudes of cell types. Exocrine cells include salivary mucous cells, sweat cells, and Von Ebner’s gland cells, which wash your taste buds. Cells in the stomach lining are different from the cells in the uterus. Hormone-secreting cells in the anterior pituitary are different from the cells in the intermediate pituitary, which are different from the cells in the gut and respiratory tracts. And thyroid cells, sensory neuron cells, central nervous system cells, and hundreds more—all containing DNA of various kinds.
In each cell, there are usually two copies of nuclear DNA (N DNA), which contains the most plentiful and precise information. The nucleus is the control center of the cell—its brain, as many scientists see it.
But here’s the rub. In his article “DNA in the Trenches,” Jonathon Jarry wrote: “N-DNA rapidly degrades beyond recognition after death. Molecular ‘scissors’ known as ‘nucleases’ are released within cells and start chopping up the DNA; bacteria, fungi, and insects have a field day with the remains; and ultraviolet radiation from the sun introduces bends and kinks in the DNA molecule which will cause problems for scientists down the road.”
This is where mitrochondrial DNA (mtDNA) comes in. “If no nuclear DNA has survived the harsh environmental conditions of a Vietnamese jungle for 40 years,” Jarry wrote, “mitochondrial DNA can become a useful piece of the puzzle.” Everyone has mtDNA, although only mothers pass it to offspring. There are one hundred to one thousand copies of mtDNA chromosomes in each cell. That means more material for identification. “Another advantage of the mtDNA genome is that it is circular,” Jarry wrote. “While N DNA is a linear molecule in an open spiral, mtDNA closes in on itself in a circle, which makes it hard for molecular scissors to chew it up. MtDNA is so resistant to degradation, it is used to identify dinosaurs.”
Not only is mtDNA an inviolable sphere, it is contained in an oblong structure with double-membrane walls that protect it from the molecular scissors. Within this structure is a sort of open maze with walls called cristae. This maze, which differs in shape for each type of cell, creates more surface area which creates more space for the multitudes of mtDNA. Within the cristae is a matrix which holds all those copies of mtDNA, tiny spheres, always active, always moving, even changing shape. While N DNA may be the brains of the outfit, mtDNA is its powerhouse.
Within each cell are other “machines.” All of them are held in suspension in the cytoplasm, a gel-like substance. Break open an egg. The yolk is the nucleus; the white is the cytoplasm. Cytoplasm holds myriad components, or organelles, that have little to do with DNA identification—Golgi’s apparatus, rough reticulum, lysosome, ribosome, and many more. Because cytoplasm is not solid, components can move around and even be exchanged with other cells because the membrane of each cell is porous. Cytoplasm is composed mainly of water but also contains enzymes, salts, organic molecules, and those organelles.
What does DNA look like? It looks like ladders. If you wind wire around a cylinder and then pull the cylinder out, the wire gives you a helix, that is, a spiral. DNA is a double helix, with two strands separated by bars, like rungs on a ladder. Life is efficient. Spirals, like mazes, take up much less room than straight strands. The “cylinder” in this case is called a histone, made of proteins, which causes the spiral to hold its helixal shape.
Without histones, the unwound DNA in chromosomes would be very long—a length to width ratio of more than ten million to one in humans. “If you stretched the DNA in one cell all the way out, it would be about two meters long,” claims BBC Science Focus.
Sugar and phosphate make up the sides of the ladder of the DNA double helix. These sides are acids. Each rung of the ladder is composed of deoxyribose (a sugar) phosphate, oxygen, and a nitrogenous base. The bases are adenine (A), cytosine (C), guanine (G), and thymine (T). Two bases in every rung connect in the middle. The rungs are held together with hydrogen bonds. But the bonding of the two bases is not random. They are paired in this way only: A pairs only with T; C pairs only with G, but the pairs can be in any sequence—AT or TA or TT and so on; GC or CG, CC, GG, etc.
ATCGTT might instruct blue eyes, while ATCGCT may instruct for brown eyes. Here is where the nucleases work, whether it is to begin replication of DNA or to destroy it after death. The rungs of the ladder are cut in the middle, separated like a zipper, disconnecting AT and CG.
The National Institutes of Health explains chromosomes: “In the nucleus of each cell, the DNA molecule is packaged into thread-like structures. Each chromosome is made up of DNA tightly coiled many times around proteins called histones that support its structure.” Chromosomes are units of all the information for cells: how to grow, to reproduce, to flourish.
Genes are portions of DNA in particular patterns. According to the National Human Genome Research Project Institute at NIH, “Every person has two copies of each gene, one inherited from each parent. Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people.” A full set of chromosomes, twenty-two pairs, contains all the inheritable traits of an organism. All it takes to make us individual is for some of those ATCG letters to be arranged differently. Otherwise, we—including animals and even some plants—are alike in most ways.
Genes control traits. Most genes, 99.9 percent, are the same in all of us. We have two eyes, a mouth, nose, hair, and the rest. Alelles are variations of genes—small mouth, large mouth, long nose, short nose, black hair, blond hair. Genes call for earlobes, for example, but alelles make them different. Some are dominant, some are recessive. An earlobe that isn’t attached to your jaw is dominant; one that is attached is recessive.
Brightstorm, a teaching website, uses the example of shirts. Let a shirt be a gene. Its color and shape are due to alelles. One shirt’s alelles call for a blue; another shirt calls for green. We inherit genes, half from Mom, half from Dad, but the combination of what is inherited is usually different for each person. Siblings can be so different as to look like neither parent because of the combination of particular chromosomes and the alelles they have inherited.
SHORT TANDEM REPEATS
Since we are all so nearly alike and alelles make us each different, the alelles are looked for to identify people. The letters of DNA are repeated hundreds of times—the GCTA, the bases or rungs of DNA. There are two to ten base pairs in a sequence; that is, the small areas of our gene sequences that are different repeat themselves in units six to twelve times. These are called Short Tandem Repeats, STRs. They alleviate the necessity of reading the entire DNA sequences, especially for remains that are old and destroyed by time and climate.
N DNA is the strongest identifier because it carries the most complete information. It is great good luck if skull bones, long bones of legs, or ribs are sufficiently intact to extract DNA. Those DNA samples are then compared to those of relatives.
How is nuclear DNA extracted from the cells? With detergent, salt, alcohol, and a centrifuge machine. Lysis, the breakage of cells, means the membranes of the outer cells and of the N DNA are broken down in order to get at the cytoplasm and the DNA inside. This is done with detergent, which separates the lipids. It works the same way that detergent washes dishes: The detergent binds to fats, which are then washed away with water. Lysis also can be achieved by grinding the material in a high-powered blender.
The next step is to add a highly concentrated salt solution. It causes fats, proteins, and other debris to form solids and fall out of the solution during the centrifuge process, forming pellets that fall to the bottom of the tube. Dissolved DNA is then moved to a new tube, and the pellets are left behind. The histone proteins, the “cylinder” holding the DNA double helix together, are removed with an enzyme called a protease.
DNA is soluble in water but not in alcohol. Rubbing alcohol or ethanol can be used, causing the DNA to clump into a visible white precipitant. The precipitant is accumulated into pellets by the centrifuge. When alcohol is removed, pure N DNA remains.
Extraction of mtDNA is more difficult. The mtDNA are far more numerous than N DNA but also more resilient. Despite the presence of multiple mitochondrial genomes in each cell, mtDNA only contains a small portion of total cellular DNA, so mtDNA samples have to be enriched before sequencing. Current extraction protocols can produce enough mtDNA to reliably sequence mtGenome from two centimeters of a hair shaft.
Current methods for enrichment either require special application of relatively expensive kits or PCR amplification of mtDNA from total cellular DNA. Polymerase chain reaction (PCR) is a common laboratory technique used to make millions of copies of a small number of molecules. This most commonly used method is relatively cheap and efficient, but a high number of PCR amplification cycles are often needed. Unfortunately, this also can lead to misinterpretation of results and, ultimately, incorrect conclusions.
DNA analysis was touted as foolproof evidence when first introduced in the early 1980s. Television shows such as CSI and Law and Order popularized DNA testing and made it seem to be the consummate truth test. “Promoters of forensic DNA testing have, from the beginning, claimed that DNA tests are virtually infallible. Although generally quite reliable, DNA tests are not now and have never been infallible. Errors in DNA testing occur regularly,” says William C. Thompson, a science writer for Gene Watch, a group that monitors developments in genetics technology.
Incorrect handling of samples can introduce accidental transfer of cellular material or DNA. Samples are collected in the field, where conditions are not ideal. Potential problems include an accidental touch by a worker, dust from wind, or animal remains in the same area. Incorrect labeling by labs and mishandling of samples can result in false conclusions, as can typos or other incorrect information on labels, or storing samples at temperatures above minus 112 degrees Fahrenheit.
People who happen to share the same DNA profile but with different alelles can be confused—especially when the comparison is based on partial DNA profiles. “Limited quantities of DNA, degradation of the sample, or the presence of inhibitors (contaminants) can make it impossible to determine the genotype at every locus,” Thompson says. In some instances the test yields no information about the genotype at a particular locus; in some instances one of the two alelles at a locus will ‘drop out’ (become undetectable).” Because partial profiles contain fewer genetic markers (alelles) than complete profiles, they are more likely to match someone by chance. The probability of a coincidental match is higher for a partial profile than for a full profile.
Comparisons are made of a person’s DNA with a sample from his hairbrush, say, or a toothbrush, teeth, or bones, as well as from genomes from relatives. Researchers are looking for similarities in the alelles. It isn’t uncommon to find similarities in one or two pairs of sites along the chosen locations of DNA among unrelated people. A match so small is only suggestive, probably a coincidence. A match of two sites makes a one in one hundred match, still room for error. But a match of thirteen sites is one in a trillion.
CONFUSION IN THE FIELD
Remains are deeply damaged after fifty years in wet, hot Southeast Asia, and they are sometimes commingled. Sometimes several bodies were left together in a battlefield. One of the strong components that helps guide researchers is personal property and personal locations, such as dog tags or a helmet with writing on it.
Still, this is not enough. Soft tissue has long since disappeared. Most of the bones have decayed, animals have carried them off, or floods have spread them over large areas. Microbes have broken down remains. DNA nucleases have destroyed nuclear DNA in the tissue and bones.
Long bones—that is, thigh bones—are best for finding DNA. Ribs are good, as are skulls. Teeth are almost as good as bone. Dental X-rays, tooth shape, fillings, implants, surgeries, and crowns are unique to individuals and can be of great use in confirming identification.
According to Indian Journal of Dentistry, “Dental tissue is amongst the hardest structures in the human body and is resistant to a number of adverse conditions such as incineration, immersion, trauma, mutilation, and putrefaction. Due to its durability and ability to withstand very high temperatures without appreciable loss of microstructure, it can survive long after the soft and skeletal tissues have been destroyed. Furthermore, because of their impervious nature, they serve as an excellent source of DNA.”
In order the handle the large numbers of missing in Vietnam, Laos, and Cambodia, as well as remains from other war zones, the Defense POW/MIA Accounting Agency (DPAA) was created. Its facility in Hawaii has the largest and most diverse forensic skeletal laboratory in the world, the Central Identification Laboratory, with connected facilities in Laos, Thailand, and Vietnam.
Information is gathered by DPAA. Additional information is submitted by veterans, families, private citizens, and amateur researchers. Every case has a “loss incident file” which includes historical background, military medical and personnel records, unit histories, official correspondence, maps, photographs, and other evidence.
That is only the beginning. After this data is assembled, teams of investigators travel to possible sites. Four to nine people in each team include medics, analysts, communication technicians, linguists, and a team leader. Some teams also use explosive ordnance disposal experts, forensic photographers, and anthropologists.
DPAA says that at least a thousand cases are being investigated at any one time. In 2016, DPAA made 164 identifications. In September of 2017 retired Air Force Maj. Gen. Kelly McKeague was sworn in as director of the DPAA. He attributed the substantial increase of MIA identification in 2016 to “talented and dedicated subject matter experts; advanced scientific methods; and a vigorous operations pace for field activities and disinterments.”
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