A geneticist’s joke begins: “How would you describe a cat?” The answer, “ACATACATACAT . . . ,” is becoming more and more a reality. As we mentioned earlier, DNA technology has allowed us to accomplish many age-old goals by new and improved means. By remembering that phenotypes, whether human, bacterial, or even viral, are the result of specific sequences of DNA, we are now at a point where knowledge of DNA can be even more effective than observing phenotypes to differentiate among organisms and people. Additionally, DNA can be used to “see” the phenotype of an organism or person no longer present, as when a criminal is identified by DNA extracted from a strand of hair left behind at the crime scene. Finally, possession of a particular sequence of DNA may indicate an increased risk of a genetic disease. Detection of a specific and unique region of DNA, or marker, can identify a per son as being at increased risk for cancer or Alzheimer’s disease long before symptoms arise. The ability to detect diseases before symptoms arise is especially important for conditions such as cancer, where early treatment can make the difference between life and death. With examples like this in mind, let’s look at several ways in which new DNA technology offers extraordinary glimpses into the present, past, and future.
DNA Profiling: A Unique Picture of a Genome
Although all DNA is based on a structure of nucleotides, the exact way these nucleotides are combined is unique for each organism. Such genetic variations, or polymorphisms, will vary to some extent even among close relatives (except identical twins, whose DNA comes from the same fertilized egg). A major goal of DNA technology is to discover these unique qualities, emphasize their differences, and provide a pattern for comparison and identification. DNA profiling (sometimes called DNA fingerprinting or typing) was developed as a forensic* technique in the mid-1980s by English geneticist Alex Jeffreys. The first methods of analysis used restriction enzymes to cut DNA located at specific recognition sites and to target genetic polymorphisms called RFLPs, introduced earlier in the chapter. The results were rendered into a series of bands or “barcodes” that could be used to compare several samples alongside each other. It turns out that hidden within genomes are large numbers of other types of DNA variations that can be used in identifying individuals that are even more specific and widespread than RFLP fingerprints.
Several of the methodologies are essential to creating a DNA profile. This includes PCR amplification for increasing the number of copies of a certain gene, electrophoresis to separate DNA fragments, and hybridization probes or primers that link to specific nucleotide sequences. The primary idea is to distinguish one isolated piece of DNA from another by comparing the genetic variations at a specific location, or locus. Note that DNA profiling is not the same as whole genome sequencing but instead focuses on a few selected genetic loci to produce a “snapshot”of a particular polymorphism pattern. Millions of such differences are known to exist throughout the genome. Many of these polymorphisms are located in regions of the DNA that do not code for protein and are considered normal rather than harmful variations.
The most reliable methods of DNA profiling presently available are (1) short tandem repeats (STRs), relatively short fragments of nuclear DNA that are covered below in some detail; (2) mtDNA or mitotyping to identify the DNA found in mitochondria; and (3) single nucleotide polymorphisms (SNPs) that locate variants of a single nucleotide in the population.
STR Profiling
The most routine methods in forensic identification are based on short tandem repeats. These are segments of DNA that consist of a short chain 2 to 8 nucleotides long. These STRs are repeated in series multiple times. Both the number of nucleotides in a sequence and the number of repeats of it vary throughout the genome. An example of this type of sequence in which four bases (GATA) re peat five times would read:
AGCT|GATA|GATA|GATA|GATA|GATA|GGCC
Studies have identified thousands of these repeated sequences, which provide the background data for producing a profile. Almost all of them occur in noncoding regions of the genome, but they are inherited the same way that genes are—as a pair of alleles, one from each parent. The genotype of a given allele can be the same or can be different, just as for eye color or blood type. At the heart of STR analysis is the pattern of nucleotides and number of repeats detected. This form of DNA profiling is mostly conducted by specialized machinery that is computer-driven and computer-analyzed, as outlined in figure 1 and discussed below.
Process Fig1. Capillary electrophoresis to detect short tandem repeats. (1) DNA is extracted from tissues, amplified using PCR, and labeled with a fluorescent molecule (note that one allele has 9 repeats while the other allele has 13). (2) The amplified DNA is added to an electrophoresis capillary. DNA moves from the negative to positive end of the capillary, with small fragments of DNA moving faster than larger fragments. (3) A laser excites the DNA molecules, causing them to fluoresce, and allowing them to be detected. (4) Information on size and fluorescence is fed to a computer, which produces an electropherogram. (5) A single peak means that a person is homozygous for the STR while two differently sized peaks means the person is heterozygous. Here, the person being tested is homozygous for the CSF1PO STR (note the single peak with 12 repeats) and heterozygous for the other four STRs (note that the alleles for the D16S539 STR have 9 repeats and 13 repeats). The numbers at the top of the electropherogram (80, 160, 240 . . .) are DNA sizes in base pairs. Barry Chess/McGraw Hill
A sample of DNA is isolated from a person or object and tagged with a number of known STR-specific primers to ready them for PCR amplification. These primers not only complement a known sequence of DNA, they also carry fluorescent dyes for later detection. After these sequences are amplified, they are electrophoresed and scanned by a laser that detects the fluorescent pattern of the primers and thus the specific STR alleles. The resultant readout, called an electropherogram, is a series of colored peaks that coincide with the STRs from that sample (figure 1). This pattern undergoes further analysis to indicate possible matches and a mathematical probability of the pattern being anyone other than the in dividual from the sample. The database contains a cross reference of the frequencies of the combinations of STRs from real populations for accuracy in comparisons.
Let us examine how this analysis would work: Imagine that a sample of blood found at a crime scene is thought to have come from a suspect and has tested positive for STR markers (alleles) A, B, C, and D. If marker A is normally present in 1 person out of 80, marker B in 1 person out of 60, marker C in 1 person out of 45, and marker D in 1 person out of 70, the probability that a single person would have all four markers is (1/80 × 1/60 × 1/45 × 1/70) or 1 out of 15,120,000. When the suspect’s markers match those seen in blood from the crime scene, it is a powerful indica tor that the suspect was involved in the crime, especially when supported by traditional evidence, such as the suspect’s having been seen near the crime scene. Adding even more STR loci can increase the probability to as high as 1 out of several billion or trillion, solidifying the match to an undeniable degree.
DNA profiling has been widely used to convict or exonerate many thousands of accused defendants in criminal cases. Besides actual fingerprints, criminals often leave other evidence at the site of a crime—hair, skin, semen, blood, and saliva. In fact, the technology is so reliable that many older cases for which samples are available are being reopened and solved. The FBI has established a national database (the Combined DNA Index System or CODIS) to support this effort. The CODIS technology analyzes 20 common STRs. This provides a uniform source for comparisons across law enforcement and forensics. Present laws require that certain offenders—exactly who varies from state to state— have their CODIS profiles entered into this system to keep tabs on known criminals.
The most frequent uses of DNA profiling are in forensics, identifying missing persons, detecting genetic diseases, determining parentage, analyzing the family trees of humans and animals, tracing the lineage of organisms, and identifying microorganisms. In a stunning application of DNA profiling, forensic scientists were able to positively identify the Unibomber, Ted Kaczynski, by DNA recovered from the back of postage stamps he had licked. This same technology has also been effective in identifying the remains of astronauts from the shuttle Columbia, some victims of Hurricane Katrina, nearly 2,000 individuals from the World Trade Center attack, and even to verify that the person killed in the raid on a Pakistani compound in 2011 was indeed Osama Bin Laden. This sort of identification of unknown DNA always requires a sample of the known DNA of the suspected person or close relatives who would share some of the STR alleles.
Mitotyping
Recall that only eukaryotic organisms have mitochondria and that each mitochondrion is self-sustaining and carries its own chromosome. Mitochondrial DNA (mtDNA) consists of about 16,500 base pairs that code for 37 genes. One advantage of mtDNA is that it is highly conserved in evolution and does not change greatly over long periods of time. Even with this small genome, it has a number of unique variations that are sufficiently varied to identify humans, plants, and animals to the level of species and even individuals. Another advantage of mtDNA is that it does not break down as readily as chromosomal DNA and remains intact even after being exposed to harsh conditions for thousands of years. Also, it is inherited only from the mother’s egg, because sperm do not contribute mitochondria during fertilization of an egg. This means that children from the same mother will have the same mtDNA profile. MtDNA was integral to the identification of the Romanovs—the family of the last czar of Russia, who were assassinated and buried in a mass grave in 1919. It has also become a tool of geneology as a way to trace the maternal lineage of families and to clarify cases of maternity.
Because even fossilized or ancient DNA can remain partially intact, ancient DNA is being studied for comparative and evolutionary purposes. Anthropologists have traced the migrations of ancient people by analyzing the mitochondrial DNA found in bone fragments. Evolutionary biologists have been able to recover Neanderthal mtDNA and use it to show that these ancient primates were in a line of evolution separate from the modern human line. Biologists studying canine origins have also been able to show that the wolf is the ancestral species for modern canines.
Several large databanks are currently collecting genomic pro files on mtDNA markers for thousands of species of plants, animals, and fungi. Two of these—Genbank and Barcodes of Life—offer these sites as a way to keep track of biodiversity (the varieties of life on earth) and to provide information for researchers and commercial operations needing to identify specimens of fish, invertebrates, plants, and other organisms that cannot be identified by the usual methods.
Miscellaneous DNA Analysis Single nucleotide polymorphisms, or SNPs, are the most specific polymorphisms because they are the most variable, occurring around 10 million times among a person’s 3.2 billion base pairs. They appear as single base differences at random sites throughout the chromosomes, both in genes and in noncoding DNA. SNPs are a valuable tool in disease diagnosis, population genetics, personalized medicine, and biomedical research. Ancestry.com and 23 & Me analyze about 700,000 SNPs when you send them a sample. More and more, in fectious disease laboratories are employing DNA test procedures based on SNPs to identify bacteria. Other forms of DNA typing are rapidly becoming the method of choice to identify pathogens, including Neisseria gonorrhoeae, Chlamydia, the syphilis spirochete, Staphylococcus MRSA, Mycobacterium tuberculosis, and a number of viruses. It is also an essential tool for determining genetic similarities between microbes involved in disease outbreaks.
Determining Gene Activity with Microarrays
Having a DNA profile or a sequence of a genome is really only half the battle. With very few exceptions, all cells in a multicellular organism contain the same DNA, so knowing the sequence of that DNA, while certainly helpful, is of very little use when comparing two cells or tissues from the same organism. Recall that genes are expressed in response to both internal needs and external stimuli and that although the DNA content of a cell remains constant, the mRNA (and hence protein) content at any given time provides scientists with a profile of genes currently being expressed in the cell. What truly distinguishes a liver cell from a kidney cell or a healthy cell from a diseased cell is their manner of gene expression.
Twin advances in biology and electronics have allowed biologists to view the expression of genes in any given cell using a technique called DNA microarray analysis. Prior to the advent of this technology, scientists were able to track the expression of at most only a few genes at a time. Given the complex interrelation ship that exists between genes in a typical cell, tracking two or three genes would be of very little practical use. Microarrays are able to track the expression of thousands of genes at once and are able to do so in a single efficient experiment. Microarrays consist of a “chip” (made of glass, silicon, or nylon) that carries bound sequences from tens of thousands of different known genes. A solution containing fluorescently labeled cDNA, representing all of the mRNA molecules active in a cell at a given time, is applied to the chip. The labeled cDNA is allowed to hybridize with any complementary DNA bound to the chip. Bound cDNA is then detected by exciting the fluorescent tag on the cDNA with a laser and re cording the fluorescence with a detector linked to a computer. The computer is programmed to interpret this data by highlighting what mRNAs are present in the cell under a variety of conditions. Look at figure 2 for an example of results comparing a normal cell with a cancer cell.
Fig2. Gene expression analysis using microarrays. Microarrays display large numbers of genes being expressed at the same time in the same cell. Small slides or chips are coated with fluorescently labeled genes and exposed to unknown cDNAs. A laser excites the bound cDNAs, while a detector records those spots that fluoresce. The color of each spot reveals whether the DNA on the microarray is present in the normal cell, the cancer cell, both cells, or neither. Darryl Leja, NHGRI
Microarrays permit the development of extraordinarily sensitive diagnostic tests that search for a specific pattern of gene expression. As an example, being able to identify a patient’s cancer as one of many subtypes (rather than just, for instance, breast cancer) will al low selection of a cancer drug that appears to be most effective. Again, we see how genetic technology can greatly assist in medical decisions.
