Two short regions of a nalurai doublc-siranded section of DNA separated by 350 bases shown as dots.
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A C-C-A-T-0 A-A-C-T-C-T-O-O-C-C-C-C-O 0-A-G-0-T-C-G-C-C-C-A-G-G-A-A-C-T-A-0-G-T-A-C-A-A-A-T .
T- O-G-T-A- C-TT-O-A-O-A-C-C -0-G- OO-C C-T-C-C-A-G-C-O-G-O- T-C-C-T-T-«-A-T-G-C-A-í-O-T-T-T-A.
. , r.-C C-A-T G-A A C T C lD GC C C C Q A-O-G T-C-G-C C-C-A-G-G-A-A-C-T-A-G-O-T-A-C-A-A-A-T. .
After adding a primer and fast - cooling
. . T-G-G-T-A-C-T-T-G-A-GAC-C-GG G C C C-T-C-C-A-G-C-G-G-G-T-C-C-T-T-G-A-TC-C-A-T
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Figure 1.4 Polymerase Chain Reaction. A. Beginning of a PCR reaction. The top frame depicts a short region of a much longer double-stranded DNA with middle piece of 350 bases represented by dots. When heated, weak bonds between the two strands are broken and the strands separate as shown in the middle frame. When the solution is fast-cooled, primers in the solution bind to the complementary sequences as shown in the bottom frame. Because the primers in this example are 19 and 21 bases long and are separated by 350, this set of primers will make a DNA product that is 390 base pairs long. B. A photograph of the result from a PCR reaction. The DNA runs in a gel from the top toward the bottom during the application of an electric current. 1: DNA size markers with the largest DNA pieces towards the top. 2: PCR product. C. The first three cycles of a PCR reaction. The original DNA is shown in dark gray, the primers in black, newly synthesized DNA in light gray. Each step first involves heating to separate the double strands of DNA, then fast-cooling to allow primers to bind, and finally allowing the DNA polymerase to synthesize a new strand ofDNA off of the primer. Dotted lines after the product of cycle 1 shows how the product of one cycle provides the template for the next cycle. The first cycle results in two double-stranded DNA, each composed of an original and the new, second, shorter piece with the primer at one end. The second cycle results in four double-stranded DNA; two are like those after the first cycle. The other two strands are both newly synthesized DNA extended from primers. Because two of these new strands are made off of a strand ending with the other primer, their lengths are determined by where the primers bind. The third cycle, shown only as the final products, results in eight double-stranded DNA molecules; two
are similar to the results of the first cycle; four are like those just described for cycle 2; and finally, the lengths of two full pairs of strands are defined by the two primers. With each successive cycle after this, the amount of double-stranded DNA doubles. Thus this reaction is called polymerase chain reaction. As the cycle number increases, the amount ofDNA defined by the two primers increases. This chain reaction allows one to make a great deal of DNA of a specific size and visualize it on a gel.
under those circumstances; once a DNA double helix is completed, the system stops because the DNA is now double-stranded and lacks regions that are partially double-stranded, the necessary condition for DNA synthesis.
This is where Mullis's creativity came to the rescue. He figured that if more primers were available, and if one could separate the two newly formed DNA strands by heating after the first round of replication and then cool the solution rapidly, the newly synthesized DNA would make new bonds with more primers. The DNA polymerase would then make more DNA by using the partially double-stranded regions formed by the primers. Thus by repeating the cycles of heating and cooling, one could make a lot of DNA identical to the DNA that began the process. There was one big problem, however. The DNA polymerase enzyme used to replicate DNA, like most enzymes, was completely destroyed by the heat necessary to separate the double-stranded DNA molecules. Fortunately, biologists had discovered, practically at the same time, that DNA polymerase extracted from the heat-loving bacteria found in hot places did resist heating very well. These bacteria live in extreme environments, such as boiling volcanic fumaroles in Yellowstone Park and midoceanic hydrothermal vents.
This is how PCR works: DNA to be copied is mixed with two short, complementary, single-stranded primers, along with nucleotides and heat-resistant DNA polymerase. The solution is heated to separate the DNA strands and cooled rapidly to allow the primers to bind. DNA polymerase then copies the primed DNA strands once. After a few minutes, the mixture is again heated so that the DNA separates into strands, and these are again fast-cooled to allow primers to bind. A second round of replication has taken place. After a few hours, the original piece of DNA has been copied thousands of times. This process is called the polymerase chain reaction because at each cycle, the number of DNA molecules is doubled.
The size of the piece that is copied is determined by the region of the original DNA to which the primers are complementary. Since the majority of the product of PCR matches the size of the DNA bracketed by the primers, this piece can be visualized using gel electrophore-sis. Gel electrophoresis is a common way to study DNA. It is called "gel" because a thin sheet of Jell-O-like medium is used to separate DNA by size, with the smaller size DNA moving faster while the larg er pieces move more slowly. It is called electrophoresis because the DNA moves through the medium due to an electric current run between the ends of the gel. We can then see the DNA pieces by using a stain.
The PCR technique can be used to amplify trace amounts of DNA from drops of dried blood, saliva, or a single hair follicle. It is also used to make DNA from scarce or degraded material, for example, mummies or fossils.
We will never forget the horrible events of September 11, 2001, that took place in New York City and Washington, D.C., in which close to three thousand innocent victims were blindly massacred. Yet, thanks to the science of genetics, many surviving relatives have had the solace of knowing that their loved ones' remains were identified. This may bring to many some sense of closure. You probably heard on TV that friends and families of the victims were requested to provide hair- and toothbrushes known to belong to those who died. This is because DNA recovered from a single hair follicle or the very few cells present on a toothbrush can be used to type a person, to obtain this person's "DNA fingerprint."
There is not enough DNA in a hair follicle or a few cells to allow direct genetic typing. However, we have seen that the PCR reaction can amplify DNA samples tens of millions of times. This is what was done in the case of the 9/11 victims where, in many instances, only body parts could be recovered, making other types of identification impossible. DNA was first isolated from hair follicles, for example, and subsequently amplified by PCR with primers known to correspond to extremely variable regions of the human genome. Human DNA contains extensive stretches that do not carry genes. The lengths of these regions vary greatly from individual to individual. Yet these stretches are flanked by other sequences that do not vary much. The principle here is to use primers that bind to the conserved regions and amplify the regions of variable length. This can be done with several sets of primers that amplify a number of different variable regions. The amplified DNA is then characterized by gel electrophoresis.
Box 1.1 continued
Figure B.1.1 DNA Fingerprinting. Gel electrophoresis of DNA samples from four individuals, represented by lanes 1-4, amplified using sets of PCR primers to four variable regions in the DNA, labeled here as A, B, C, and D. The lanes labeled L provide identical reference markers used to calibrate the distances that the DNA bands traveled in samples 1-4. Note that only one single band in D is shared by all four individuals.
Next, DNA was extracted from the re-~ ~ — mains of the victims and processed in the ™ — ^_ — same way. By comparing the length of am-c 2 plified DNA from the remains with those
" — — from the hairbrush, it is possible to provide positive identification of the victims (see „ figure B.l.l). This application shows that _ ** genetic technology is now indispensable to
— — — solving forensic problems. The same ap-
m. — — -■ *" proach is used in paternity cases.
_ Another example of DNA typing is of his-
_ — torical importance and has helped uncover tm — ~ an impostor posing as a member of an imperial family. In 1918, Nicholas II Romanov, the last tsar of Russia, his wife Alexandra, and their children were assassinated by Bolshevik revolutionaries. Their bodies were dumped into a shallow unmarked grave. In 1993, their bones were dug up and identified by DNA typing. This was possible because Prince Philip of Edinburgh, the husband of Queen Elizabeth II , is a relative of the deceased Alexandra. Their DNA profiles matched.
Interestingly, shortly after the assassination took place, rumors started circulating that one of the daughters of Nicholas II and Alexandra, Anastasia, had survived. In 1922, a woman claiming to be Anastasia surfaced in Berlin and was able to convince many émigrés of the Russian nobility that she was indeed Anastasia. Some, however, were not convinced. Later, this woman emigrated to the United States under the name of Anna Anderson. She died in 1984.
Thanks to preserved tissue samples that were kept in a hospital where she had undergone surgery, her DNA could be typed. The results were negative; Empress Alexandra and Prince Philip were not related to her. Why Anna Anderson claimed to be Anastasia is unclear. She maintained till her death that she was a Romanov. However, she never benefited, monetarily or otherwise, from her lies. We know now that she was simply an impostor.
We have learned in this chapter about the chemical composition of DNA, its double-helical structure, and its role as genetic blueprint. We also can see from its double-helical structure, based on complementary base pairing, how DNA can be copied. This understanding of DNA replication has led to the discovery of a technique, PCR, that allows the production of substantial amounts of DNA from very small amounts. PCR is now used on a routine basis in laboratories doing basic research and in forensic laboratories.
Try This at Home: Extract DNA from Vegetables in Your Kitchen
Have you wondered what DNA looks like? It is fairly easy to extract DNA using common equipment and materials found in your kitchen. The following is one recipe for isolating DNA. The recipe mentions onion but you can use other vegetables, such as lettuce or celery.
1 small onion meat tenderizer dishwashing detergent cheesecloth denatured alcohol (can be purchased at a pharmacy)
Try This at Home continued
Peel and chop up 1 onion and place in blender.
Add twice the amount of water and blend until fine.
Add 1-2 tablespoons dishwashing detergent. This is to emulsify the membranes around cells that are made of lipids.
Add 1 tablespoon meat tenderizer. Meat tenderizer is typically made from papaya or other fruits that contain protease, an enzyme that breaks down proteins. This helps to release the DNA from proteins.
Gently (so that the mixture does not become foamy) mix in the detergent and meat tenderizer.
Filter through cheesecloth to get rid of plant debris.
Gently layer cold denatured alcohol on top of the clear filtered juice.
The white material at the interface is DNA!
If you want to collect the DNA, try spooling it up with a chopstick.
HUMANS HAVE TINKERED WITH PLANTS and animals since before the dawn of recorded history. For example, it is thought that dogs were domesticated from wild wolves about 15,000 years ago. All the crops we eat today, including wheat, rice, and corn, are the products of thousands of years of breeding for larger grains, edibility, flavor, and other desirable characteristics. In addition to the domestic dog, horses, cows, goats, and pigs were bred from their wild ancestors. Obviously, domestication was a great success, for we now have many varieties of these useful crops and animals suited for different parts of the world and used for different purposes. Yet, despite this obvious success, until about a hundred years ago we did not know how inheritance worked. Breeders could generally state that many traits appeared to be inherited. But when it came to details of that inheritance, some traits seemed to be blends of features from both parents, whereas other traits appeared to copy the trait from only one parent. Sometimes traits from grandparents not expressed in the parents reappeared. Occasionally some useful traits seemed to arise completely anew! How were breeders to make any sense of these observations?
Breeders had trouble understanding how inheritance worked because many traits represent the manifestation of many genes and can also be affected by the environment. Many traits of great interest fall into this category, such as the amount of milk produced by cows, the size of a fruit, the amount of oil in canola seeds, the color of our skin, and our propensity for high blood pressure, just to name a few. We will look into these more complex traits determined by many different genes in chapter 12.
Plants Are Good Organisms for the Study of Inheritance
Near the end of the nineteenth century, many plant breeders were keenly interested in how inheritance worked. They began to realize that what was needed was a systematic study of hybridization, or crossbreeding, between closely related plants possessing obvious differences in a single trait or just a few traits. Fortunately, a serendipitous choice of traits that were under the control of single genes enabled them to make sense of inheritance. Gregor Mendel was the first to figure out how inheritance worked for single-gene traits. His careful choice of plant, peas, and their character, as well as his statistical analysis of many offspring, revealed the bases of inheritance. But his discovery went unappreciated until 1900, long after his death in 1884.
Because many of the early studies of inheritance used plants, let's take a few moments to learn a bit about plant biology and reproduction. Most plants, unlike animals, are hermaphrodites; that is, one individual possesses both male and female parts. The pollen grains are like the sperm cells of the flower. They are contained in a structure called the anther. The ovule is the female reproductive cell that is found at the base of the structure called the style. This structure is topped by a sticky part called stigma that receives the pollen. The fact that plants are hermaphrodites allows scientists to perform controlled crosses that would not be possible with animals. For example, plants can be selfed, short for self-fertilized or self-pollinated. Selfed means that the male (pollen) and female (ovule) contributions to an offspring come from the same individual. Many plants are easily selfed by enclosing the flower to prevent pollen from another individual from entering the flower that contains both the anther and stigma. In order to perform crosses between different individuals, the anther is removed from the individual that will be contributing the ovule. Pollen from the anther of a different individual is picked up with a fine brush and brushed on the stigma of the first individual. This requires a bit of practice, but, once learned, it is fairly easy to do. Also, many plants produce a lot of seeds, that is, offspring, from a single cross. The ease of controlled crosses, the production of many offspring, and relatively fast generation times make many plants ideal for the study of inheritance.
Early in the study of heredity, most scientists and breeders thought that traits of offspring were due to blending traits of the parents. After all, there were clear examples of such blending, for example, skin color, the heights of many plants, and so on. This interpretation sounded quite reasonable because male and female gametes contribute to the formation of the offspring. Nevertheless, the idea of blending inheritance could not explain many features of inheritance. One common occurrence that could not be explained by blending inheritance is a trait's skipping generations. That is, a trait is present in the grandparent, not present in either parent, but appears in the offspring.
Crosses of parents that did not exhibit this blending feature were seized upon to try and understand inheritance of traits. That is, exceptions to the generally accepted idea of blending inheritance allowed researchers to ask why some traits did not exhibit this behavior. Edith Saunders, at Cambridge University, England, who later became the president of the Genetical Society, conducted extensive studies in the 1890s. She studied a number of different garden and alpine flowers. For example, she noted that there were two varieties of plants in the cabbage family of alpine plants. The plants are similar in all respects, except that one has hairy leaves while the other has smooth leaves. These plants grew right next to each other in the meadow. Saunders reasoned that if insects freely pollinated the flowers, and the traits shown by the offspring were a blend of parental traits, two distinct forms should not persist for long. So, Saunders brought these plants into her garden in order to experiment with them, under conditions where she could control their pollination. Saunders observed that by cross-pollinating a hairy-leafed plant with a smooth-leafed one, she obtained twenty-one hairy plants and ninety-nine smooth-leafed plants as the offspring (figure 2.1) and no plants with intermediate hairiness. In another example, she used a fragrant garden flower, called stock, that has very smooth leaves. Crossing this smooth-leafed plant with one covered in gray hair from the wild resulted in only smooth or completely hair-covered offspring and nothing in between.
Figure 2.1 Cross Between Smooth-Leafed Plant and Hairy-Leafed Plant. When Edith Saunders crossed smooth-leafed plants with hairy-leafed plants, she obtained only smooth-leafed or hairy-leafed plants. No partially or less hairy leaves were observed. These are images photographed through the microscope illustrate the difference between hairy and smooth leaves. In a cross between these plants, Saunders obtained ninety-nine smooth-leafed plants and twenty-one hairy-leafed plants. Photos of leaves courtesy of Valerie Lynch-Holm. The plants are from the WSU Owenby Herbarium.
Saunders interpreted her results as clear examples of nonblending inheritance. The stage was set for the rediscovery of Mendel's results.
The Dutch botanist Hugo De Vries rediscovered Mendel's work in 1900. He looked at many different flowering plants, but for each cross, he focused on only a single trait that differed between closely related plants. When De Vries crossed a plant of one type with another plant differing by only a single trait, all the resulting offspring showed the trait of one parent. For example, he crossed a red campion with a white campion. He found that the first generation seedlings all produced red flowers (figure 2.2). When he then allowed these red flowered offspring to self-pollinate, he got 73 percent red and 27 percent white, or close to a 3 to 1 ratio. He did another experiment using the same species of plants, but this time he chose varieties that differed in the hairiness of their leaves. The first generation produced all hairy plants, but a self-pollination of those hairy-leafed offspring produced 72 percent hairy-leafed and 28 percent smooth-leafed plants. Among many such experiments, he saw approximately 75 percent of one type
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