Science provides a story about the natural universe around us. It is a story that inspires wonder and, at the same time, has enormous practical implications for every aspect of human existence. As is the case for any scientific discipline, success in molecular biology is defined to a large extent by the contributions a scientist makes to the larger scientific story. That ongoing story provides a context and community within which scientists frame every experiment.

The history and philosophical underpinnings of science have brought about guidelines and rules for new experimentation at every level of this intertwined enterprise. The scientific community is highly interactive, with ongoing discussions supplying both constraints and insights that can stimulate progress. The collective discussion is carried out in informal conversations, at scientific meetings, and, most importantly, in the peer-reviewed scientific literature. A successful contribution is usually one that eventually appears in this literature.

Molecular biology is a scientific enterprise, giving rise both to information, as conveyed in the chapters of this text, and to future advances. An understanding of the scientific enterprise can help in our assimilation of existing information and accelerate success in any scientific effort.

Science is both a body of knowledge and a process for generating that knowledge. In the scientific community, scholars attempt to answer questions about the natural universe. Since the dawn of humanity, people have employed many approaches to understanding the world around them, guiding human development for millennia. The modern version of the scientific method, relying on careful observation and experimentation, has been widely applied for only about four centuries.

The scientific approach to discovery has at least three characteristics. First, science focuses only on the natural universe. The realm of science is thus limited to what we can observe and measure. Second, science relies on ideas that can be tested by experiments and on observations that can be reproduced. Finally, the experiments are carried out within an ever-expanding web of scientific theories that provide guidance and insight along the way.

Science makes one philosophical assumption: that forces and phenomena existing in the universe are not subject to capricious or arbitrary change. They can be understood by applying a systematic process of inquiry—the scientific method. The French biochemist Jacques Monod referred to this basic underlying assumption as the postulate of objectivity. Stated more simply, science cannot succeed in a universe that plays tricks on us. This assumption is made every time a scientist performs an experiment. If an experiment is repeated and the results are different, there is always a reason; something must have been different during the second experiment. When faced with seemingly contradictory results, every scientist is trained to figure out why. Often, inconsistencies have a trivial cause, such as a degraded or inactive reagent; but sometimes they lead to great insights.

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In modern science, an initial idea is called a hypothesis—a proposal that provides a reasonable explanation for one or more observations but is not yet substantiated by sufficient experimental tests to stand up to rigorous critical examination. A scientific theory is much more than a hypothesis; it provides an explanation for a body of experimental observations and is thus a firm basis for further inquiry. When a scientific theory has been repeatedly tested and validated by many types of experiments, it can be considered a fact.

Scientific hypotheses and theories can also be defined by their presence in the peer-reviewed scientific literature. Papers are accepted or rejected for inclusion in that literature based on professional reviews by other working scientists. According to the Publishers Association of the United Kingdom, there are more than 16,000 peer-reviewed scientific journals worldwide, publishing some 1.4 million papers each year. This continuing rich harvest of information is ultimately the foundation of scientific progress.

The first recorded efforts to systematize scientific inquiry have been credited to the classical Greeks, who were clearly influenced by other civilizations in Babylon, Assyria, Egypt, Persia, and elsewhere (Figure 1-13). Key thinkers replaced trial and error, chance discoveries, and appeals to the supernatural with a more formalized system of reasoning. Euclid, Aristotle, Plato, and others made significant advances by using inductive reasoning, making a broad conclusion from specific facts (e.g., my stove is hot, my brother’s stove is hot, so all stoves are hot), and by deductive reasoning, forming a conclusion from a premise (e.g., premise: all flying organisms are birds; observation: a crow flies; conclusion: a crow is a bird). Experiments played a minor role. The process worked reasonably well for mathematics and engineering, less well for most other pursuits. Scientific progress declined in the West with the decline of the Roman Empire, symbolized by the assassination in 415 ce of the last director of the great library of Alexandria in Egypt, the accomplished Greek mathematician and philosopher Hypatia.

A reawakening began with the emergence of Islamic science during the Middle Ages, building on Greek and Roman ideas with rational arguments combined more liberally with observations and experiments. The physicist Abū ‘Alīal-Ḥasan ibn al-Hasan ibn al-Haytham (965–1040), known as Alhazen in the West, is regarded as the father of modern optics. Alhazen was particularly influential in refocusing the scientific process on reproducible experimentation. Latin translations of Arabic and Greek texts filtered into Europe, helping to stimulate the Renaissance. The Franciscan friar Roger Bacon (1214–1294) was inspired by these texts. He was among the first to describe a method of inquiry involving observation, hypothesis, and experiment, while also stressing independent repetition to verify results.

By the beginning of the seventeenth century, the flaws in Aristotelian philosophy had become clear to many scholars. René Descartes (1596–1650), Galileo Galilei (1564–1642), and Francis Bacon (1561–1626) led a revolution in scientific thinking. Frustrated with the impediments inherent in the Aristotelian system (e.g., if one encounters a bat, the premise that all flying organisms are birds is falsified), they laid the groundwork for the modern scientific method.

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In 1619, Descartes set down four rules for inquiry, which we paraphrase here: (1) never accept anything as true unless that truth can be clearly demonstrated; (2) reduce a problem to its parts; (3) begin with the simplest part and gradually work to the more complex; and (4) be thorough. Francis Bacon divided inquiry into a somewhat similar series of steps, first gathering relevant observations, and then deriving increasingly complex conclusions from them. Galileo combined careful observation and rational argument to develop new fields of inquiry.

The scientific method had matured by the beginning of the nineteenth century. In its most basic form, it begins with a question about nature. Relevant observations are made, and a hypothesis is crafted to explain them. Assumptions that underlie the hypothesis are defined, and each assumption is carefully tested by experimentation. Experimental results lead to acceptance, rejection, or modification of the hypothesis. Additional experiments can lead to the acceptance of a new theory or fact about the natural world.

In modern molecular biology, this now classical version of the scientific method underlies huge numbers of advances. One example is the discovery of the DNA-synthesizing enzyme DNA polymerase by Arthur Kornberg and his coworkers in the 1950s (discussed in Chapter 11). With the elucidation of DNA structure by James Watson and Francis Crick, Kornberg and others hypothesized that an enzyme must exist to synthesize this polymer. They assumed that the enzyme could be purified and studied, and its need for a template determined. To test these assumptions, they lysed (broke open) bacterial cells, fractionated the extracts to separate the many proteins and enzymes, and developed assays to measure the activity of the hypothetical enzyme in the presence of all plausible substrates and precursors. The effort paid off in 1956, with an assay that detected the incorporation of nucleotides into DNA polymers. DNA polymerase was subsequently purified, named, and thoroughly characterized by the same team, substantiating their original hypothesis; the existence of a DNA polymerase became fact.

The path to scientific discovery is rarely as rigid, linear, and empirical as implied in the preceding paragraphs. Molecular biology is certainly replete with examples of the purposeful application of the scientific method, but it is also full of surprises, excitement, and occasional messiness. Major discoveries are often characterized by hard work, extraordinary perseverance (a surprising number of discoveries seem to be consummated in the middle of the night), and more than a little innovative thinking. Many such examples are chronicled throughout this book.

To understand how science is done, we need to expand the concept of the scientific method to include the contribution of scientific context. Scientific inquiries are not carried out in a vacuum. The discovery of DNA polymerase, described above, was much more than “hypothesize the existence of this enzyme and then go get it.” The search was carried out in the context of the ideas and facts that prevailed at the time. These included information about the structure of DNA, the chemistry and thermodynamics of enzyme catalysis, cellular physiology and metabolism, the properties of proteins, and the recent finding of a related enzyme, ribonucleotide phosphorylase, by Severo Ochoa. The entire discovery process was guided by the interconnected web of theories and information within which the DNA polymerase hypothesis was developed and tested.

To understand the scientific method, it is also instructive to examine other examples of how important ideas were conceived and tested. The examples below are meant to demonstrate the variety of ways in which science is advanced, and they underscore the fact that science is a very human experience, despite the rigor of the discipline.

Hypothesis and Discovery This classical path to scientific discovery led Kornberg and colleagues to DNA polymerase. It was also used by Jack Szostak in his exploration of prebiotic chemistry (see Moment of Discovery at the opening of this chapter), as well as by countless other scientists. Bob Lehman describes the quite similar path to his discovery of DNA ligase, an enzyme that joins segments of DNA (see the Moment of Discovery for Chapter 11). These discoveries started with hypotheses and ideas reasonably based on the knowledge of the time, and the hypotheses were carefully tested. In addition, the discoveries were made within a broader context of constraints imposed by theories related to the geologic history of our planet, basic chemistry, nucleic acid chemistry, structural biology, the properties of enzymes, and many other areas of knowledge.

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Model Building and Calculation As we will see in Chapter 6, Watson and Crick relied on intuition and important experimental clues from other researchers to build a model of DNA structure so clearly fitting the data that its acceptance was both shocking and immediate. A similar approach can be seen in the work of Steve Mayo in his efforts to deduce the three-dimensional structure of proteins from their amino acid sequence (see the Moment of Discovery for Chapter 4). In this variant of the scientific method, a researcher draws on experiments performed by others and mines the unrealized implications of that existing body of knowledge to bring about a significant advance in understanding. Insights obtained in this way are almost always subjected to further experimental examination to confirm them. In both of the cases described here, later solution of the three-dimensional structures of the macromolecules in question confirmed the results of the original investigations.

Hypothesis and Deduction Soon after the structure of DNA was determined, it became apparent that the information in DNA is converted into RNA, and then into protein. However, how amino acids could bind to RNA molecules to guide assembly of the amino acids into proteins remained unclear. Then, in 1955, Crick wrote an influential note to some colleagues that laid out his “adaptor hypothesis,” proposing that there was an as yet undiscovered molecule that linked each amino acid to sequence information in the RNA. This insight, never published but widely disseminated, was not an endpoint. The hypothesis was subjected to experimental confirmation, and the adaptor—transfer RNA (tRNA)—was discovered within a year by Paul Zamecnik and Mahlon Hoagland (see the How We Know section in Chapter 17).

Exploration and Observation Darwin’s voyage on the Beagle was not designed to answer the questions that his work eventually addressed, and no hypotheses were offered when the ship set sail. Thousands of individual observations (and context provided by the work of many predecessors and contemporaries) simply gelled in a great mind to create a transformational theory. Molecular biologists routinely embark on similar voyages, with computers and new technologies as vessels of discovery. Rather than focusing on one or a few enzymes and reactions, we can now approach issues related to entire systems. Cells, organisms, and even ecosystems are being explored. Many of these technologies are described in Chapter 8. The new technologies enable us to explore broadly, asking: what’s out there that we have missed? These efforts provide a rich context for new ideas and hypotheses.

Inspiration The polymerase chain reaction, often abbreviated PCR, is a convenient process for amplifying a DNA sample. PCR is now so integral to biotechnology and forensic science that it is hard to imagine doing molecular biology without it. PCR was invented by Kary Mullis in a flash of inspiration that came to him while driving along a northern California highway in 1983. Reflecting on one problem, Mullis stumbled on a solution to an even greater one: a method to produce almost unlimited copies of a DNA sequence in a test tube. (This story is presented in more detail in the How We Know section in Chapter 7.) Although this advance was not precipitated by an orderly application of the conventional “question, hypothesis, experiment” approach, the inspiration was a logical and probably inevitable outgrowth of the growing body of knowledge that constituted the molecular biology of that moment.

Serendipity Accidental discoveries are, by definition, unplanned and unexpected, and they can change the way we think about living systems. Such discoveries are not preceded by a carefully orchestrated quest organized around a posed question. They just happen—and they happen often. One example involves the discovery of RNA catalysis. In the early 1980s, Thomas Cech was investigating the mechanism by which segments of RNA (introns) are removed from a messenger RNA in a reaction known as RNA splicing (discussed in Chapter 16). Searching for the protein enzymes that catalyzed the process, Cech and his coworkers were startled when the splicing reaction still occurred in a necessary control trial, in which the protein extract was left out of the reaction mixture. After long and careful experimentation to eliminate any possible source of protein contamination, they were able to report the paradigm-shifting discovery of an RNA-catalyzed reaction.

These many pathways vary in detail, but they have characteristics in common that identify them as part of a modern and adaptable scientific method. They all focus exclusively on questions and properties related to the natural universe. All the ideas, insights, and experimental facts that arise from these endeavors rely on reproducible observation and/or experiment. Ultimately, the information from the various approaches fits into the web of scientific knowledge. The experimental efforts and results can be used by other scientists anywhere in the world to build new hypotheses and make new discoveries. An attempt to construct a flow chart for a realistic application of the scientific method is shown in Figure 1-14

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Any individual who uses the scientific method to answer questions about the natural universe is a scientist—from a high school student examining pond water with a microscope to an investigator using high-tech equipment at a major research institute. A specific academic degree is not a prerequisite for making a scientific contribution. However, success in science is strongly correlated with training and experience.

Scientific training is much more than lessons about experimental methods; it is an education in how to think about and approach scientific problems. Progress often demands a willingness to give up a favorite hypothesis. In his book Advice to a Young Scientist (1979), the Nobel Prize winner Peter Medawar expressed it thus: “I cannot give any scientist of any age better advice than this: the intensity of the conviction that a hypothesis is true has no bearing on whether it is true or not.”

If any idea about the natural universe does not align with reproducible observations, a scientist must challenge it. Except for the postulate of objectivity, no assumption is inviolate. The ideas that a scientist accepts must be based on evidence that can be observed, measured, and reproduced. The standard of reproducibility, coupled with the continual advance of new experimentation, provides a robust system for weeding out the inevitable errors and for guiding insightful reinterpretations.

The many decisions along any path to discovery, such as determining the number and types of tests to use in examining a hypothesis, are guided by training and experience. There is no prescribed formula for these decisions, but they are much less subjective than they might at first appear. The context of scientific theory and information plays a major role in ensuring rigor. When a DNA-synthesizing enzyme was first purified from bacterial crude extracts by Kornberg and his colleagues, specific tests had to be performed to establish that the enzyme was involved in normal chromosomal replication. These tests were dictated by the broader scientific context in which the discovery was made. The experiments were conceived, in part, by the need to fit this new enzyme into the existing web of scientific information.

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When a result contradicts the original hypothesis, a scientist must decide whether to reject or revise. This decision is similarly rooted in both the context of scientific information known at the time and the experience of the investigator. When Bob Lehman and colleagues detected the action of DNA ligase (in the bacterium Escherichia coli) for the first time, they anticipated that a high-energy source, probably ATP, would function as a necessary cofactor for the reaction. This expectation was rooted in a great deal of precedent from similar reactions. But when ATP failed to stimulate the ligation reaction, the DNA ligase hypothesis was not abandoned. Instead, the investigators questioned their assumptions about ATP. A search eventually identified another cofactor, NAD⁺, as the critical energy source for the E. coli DNA ligase, revealing a new chemical function for this important cellular molecule.

In science, the work is not done by individuals in isolation, and scientific decisions are influenced and affected by the scientific community at large. It is not enough to demonstrate a new idea to oneself. To integrate an advance into the web of scientific information, a scientist must make a case that is compelling to the broader community. A continual discussion within this community fosters progress and helps ensure the integrity of the information generated. A few years after Kornberg first isolated DNA polymerase, the involvement of this enzyme in chromosomal replication was questioned. The rate at which it synthesized DNA seemed insufficient to match the observed rate of chromosomal duplication in cells, and cells somehow survived when the gene encoding the enzyme was eliminated. These observations led to discovery of another DNA polymerase in the same bacteria, now called DNA polymerase III, that serves as the primary enzyme in chromosomal replication. The original DNA polymerase (now called DNA polymerase I) is more abundant than DNA polymerase III, hence its initial detection. DNA polymerase I has a variety of auxiliary functions in DNA replication and DNA repair, as we will see in Chapters 11 and 12.

When an idea is tested, the most compelling case is made when the idea passes multiple tests involving two or more different techniques. Collaboration among scientists often strengthens the case. Different individuals bring different perspectives and expertise to bear on a problem. The cross-talk can correct errors, provide novel insights, and make connections that one individual might miss. The case becomes even more compelling when tests are replicated in multiple laboratories. To gain entry to the scientific literature, the work must pass the inspection of fellow scientists. Peer reviews of scientific papers, and of grant applications, are sometimes challenging and even painful experiences for the authors, but they are critical for ensuring quality and rigor. A conscientious participation in this peer-review system is a shared duty of every working scientist.

Ultimately, this literature documents the scientific story about the natural universe. That story is ever expanding, never complete, and always ready for new contributors.

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