A brief history of medical diagnosis and the birth of the clinical laboratory, Part 5a--the foundation of molecular science and genetics.

CONTINUING EDUCATION 

To earn CEUs, see test on page 24.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Identify six scientists and explain how their discoveries have proven to be pivotal to the science of genetics.

2. Discuss three major discoveries in molecular biology that have influenced the development of molecular diagnostics.

3. Describe four inheritable disorders for which genetic testing is useful in risk prediction and diagnosis.

4. List three goals achieved in the Human Genome Project.

5. State three challenges to widespread application of molecular diagnostics.

**********

At the conclusion of "Fraud and abuse, managed care and lab consolidation"--Part 4 of this series of articles in December 1999--the author wrote: "Only a few years ago, laboratory visionaries predicted that developments in molecular biology had the potential to change laboratory medicine in the same way that computed tomography and magnetic resonance imaging altered the practice of radiology. Speculation that routine hospital admissions testing done in the 21st century could include a panel of DNA probes in place of a chemistry profile or complete blood cell count now look more plausible than ever.... On the verge of the 21st century, the lab is providing more information about the human condition faster and more accurately than ever. It is strategically positioned for success in the healthcare industry--in the business of supplying critical information in the information age." (1)

[ILLUSTRATION OMITTED]

Where do we stand?

It is now 2006, and the future is here. The healthcare industry is burgeoning with new companies that are involved in the molecular "revolution," many of which have come into being as recently as the completion of the sequencing of the human genome was announced on April 14, 2003. It was at this point that the so-called genetic blueprint of life was available to researchers and scientists who began the continuing mad genomic scramble, this time to identify the approximately 30,000 human genes. (2)

Biomedical research has been driven by the prospect of discovering what genes are involved in diseases as complex as cancer and diabetes. Already, new treatment methods and "designer drugs"--made to suit a particular genetic profile--are being enabled by the sequencing results, as is earlier diagnosis of certain diseases through genetic testing. In the past decade, with the availability of technology and with knowledge fueled by the investment and interest in the Human Genome project, molecular diagnostics has recently enabled laboratories to offer diagnostic and predictive tests for inherited disorders. (3) And while molecular testing seemingly arrived all in a flurry, there is a long history of unglamorous trial-and-error behind today's movement toward personalized medicine, which involves pharmacogenomics and nutrigenomics.

Mendel to Morgan to modern DNA 

The precursor to the current era of molecular genetic testing in humans reaches back 141 years to Gregor Johann Mendel's 1865 publication of experimental data. Mendel (1822-1884) joined the Augustinian Order of monks in 1843. His experiments with peapods in the monastery's garden led him to formulate the basic principles of heredity. Between 1856--three years before Charles Darwin's Origin of Species was published--and 1863, (4) Mendel cultivated and tested some 28,000 pea plants. His experiments brought forth two generalizations which later became known as Mendel's Laws of Heredity or Mendelian inheritance.

His basic tenets related to the transmission of hereditary characteristics from parent organisms to their children. When Mendel published his theory in 1865, biologists who did not believe his results were especially important largely ignored them. Even Mendel himself believed that his results applied to only certain categories of species and did not thoroughly understand his theory's applicability.

At the start of the 20th century, Mendel's work was "rediscovered," arousing much controversy. Linked in this confused rediscovery, European scientists Hugo de Vries, Carl Correns, and Erich von Tschermak brought Mendel's early theory of heredity to light. Despite a number of detractors and a few promoters, Mendel's ideas were eventually merged with Thomas Hunt Morgan's chromosome theory of inheritance in 1915 and, thus, became the core of classical genetics. (5,6)

Morgan was an American geneticist and embryologist (1866-1945). He received his bachelor's degree from the State College of Kentucky (now the University of Kentucky), his PhD from Johns Hopkins University, and at Bryn Mawr worked on embryology during his tenure there. By 1910, following the rediscovery of the Mendelian model in which the chromosomes of cells were thought to hold the actual hereditary particles, Morgan's research moved to the study of mutation in the fruit fly: Drosphila melanogaster. In Morgan's famous Fly Room at Columbia University, he demonstrated that genes are carried on chromosomes and are the mechanical basis of heredity--forming the foundation of modem genetic science

and guaranteeing Mendel's place in scientific history. (6)

The move toward revealing DNA

From this point on, many scientists not nearly so famous as Mendel and Morgan labored on with experiments that, one by one, continued to add to the body of knowledge concerning genetics. While Linus Pauling and his colleagues introduced the term "molecular disease" into the medical vocabulary in 1949 (based on their discovery that a single amino acid change at the beta-globin chain leads to sickle-cell anemia), (7) the next big public sensation in the field was the discovery of DNA by Watson and Crick. While the two men did not become household words, the three little letters D-N-A did.

Deoxyribonucleic acid (DNA) was actually isolated in 1869 by Swiss chemist Friedrich Miescher. He later demonstrated that DNA exists only in chromosomes, the site of hereditary material. By the 1930s, DNA was known to be a large molecule in the form of a long chain of nucleotides; but, other than that, its structures and functions were poorly understood. By then, geneticist George Beadle and biochemist Edward Tatum teamed up to investigate the relations between genes and enzymes. In 1943, Oswald Avery had identified the genetic role of DNA: that DNA carried genetic information and might well be the gene. (8)

Two teams, two approaches, one answer

Almost as fascinating as the scientific studies that unfolded from Mendel down to Avery is the tale of how James Dewey Watson and Francis Harry Compton Crick converged at the same time, in the same place and, in 1959, cracked the DNA code. Crick was a Briton who had studied physics, then chemistry and biology, and still had not earned a doctoral degree. At age 19, Watson--an American--had graduated from the University of Chicago and had received his doctorate at age 22. He had studied ornithology but changed career paths when he went to Europe for post-doctoral studies.

Another team--Maurice Hugh Frederick Wilkins and Rosalind Elsie Franklin--were working with DNA at King's College in London. At a conference in Italy, Watson heard Wilkins speak and was introduced by him to a photograph of a DNA molecule which had been rendered by his colleague, Franklin, via X-ray crystallography. With this bit of knowledge, Watson was immediately keen to solve the riddle of DNA. (9)

Watson (now 23, became a research fellow at Cambridge) and Crick (now 35, and a graduate student there) had admired the work of Linus Pauling who had discovered in 1948 "that many proteins take the shape of an alpha helix, spiraled like a spring coil." (8) They were also aware of the studies of Erwin Chargaff, a biochemist, who, in 1951, announced that "the arrangement of nitrogen bases in DNA varied widely, but the amount of certain bases always occurred in a one-to-one ratio. These discoveries were an important foundation for Watson and Crick's later description of DNA." (8) The two busied themselves by constructing physical models in order to create an accurate picture of the DNA molecule.

Meanwhile. Wilkins and Franklin continued experimenting with X-ray diffractions images of DNA. By using the technique, "the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam." (10) Franklin was the first to state that the sugar-phosphate backbone of DNA lies on the outside of the molecule and described the helical structure of the molecule. (10)

Watson attended one of Franklin's lectures in 1951 during which she outlined her work to date. With a rather fuzzy memory of what Franklin had described, Watson returned to the Cavendish Laboratory at Cambridge and, with Crick, constructed yet another failed model. At this point, the head of the laboratory directed them to end their DNA research. (8) That order was ignored, and the two labored on in what had become their self-generated competition with Pauling as well as Wilkins and Franklin.

Two years later, in 1951, a frustrated Wilkins--having already made Watson's acquaintance in Naples, Italy (9)--showed Franklin's results to Watson, reportedly without her knowledge or consent. The results of Franklin's work with X-ray diffractions were that DNA had all the characterisitcs of a helix. (6) With the evidence that Wilkins delivered, Watson and Crick resumed their construction of physical models, combining Franklin's and Pauling's information on helical structure with that of Chargaff's latest evidence about base pairs. By 1952, much was known about DNA. including the fact that it was the sole substance capable of storing all the information needed to create a living being. What was not yet known was what the elusive DNA molecule looked like, or how it performed this amazing hereditary function. This would change in the course of a single year. (10) Their model ended up having matching base pairs interlocked in the middle of a double helix, so that the distance between the chains was consistent throughout. (8)

DNA and the ongoing search for answers

The two eager scientists, Watson and Crick, demonstrated that each strand of the DNA molecule was a template for the other. "During cell division, the two strands separate and on each strand a new 'other half' is built, just like the one before. This way, DNA can reproduce itself without changing its structure--except for occasional errors, or mutations." (8) They published these findings in Nature in 1953; Rosalind Franklin's findings were published in the same issue as a "supporting article." (8) Because their model had fit experimental data so well, Watson and Crick's structure was widely accepted. In 1962, Watson, Crick, and Wilkins won the Nobel Prize for physiology/medicine. Rosalind Franklin had, by this time, died at age 37 of ovarian cancer, and by the conditions required by the Nobel Prize Committee that only living persons be recognized, she could not be one of the recipients. (8)

"The discovery of DNA, the explanation of its construct, has been acknowledged as the most important biological work of the last 100 years, and the field it opened may be the scientific 'frontier' for the next 100." (8) In the 50 years since Watson and Crick revealed their DNA findings, the field of molecular diagnostics has flourished (see Figure 2). The 1985 invention of polymerase chain reaction (PCR) provided the boost that scientists needed to improve their capabilities to diagnose inherited diseases on the DNA level. (9)

"PCR opened the door to eliminating complexities, costs, and time requirements of available technologies like cloning and sequencing. Its ability to generate exponential copies of a target sequence means that a known mutation can be identified within a day rather than months. PCR also eliminated the necessity for radioactivity for routine molecular diagnosis, so that the clinical laboratory is now able to provide genetic services for carrier or population screening for known mutations, as well as prenatal diagnosis of inherited diseases. From PCR, mutation-detection techniques developed can be categorized under enzymatic-based methods, electrophoretic-based methods, and solid phase-based techniques." (9)

Some of the disease-related gene mutations are recessive (they must be present in both gene copies, one from each parent) before they cause dysfunction, while others are dominant (a single altered gene copy can cause disease). Some are X- or sex-linked, associated with the X or Y chromosome that determines our gender, and are found only in males or females. Some mutations have arisen and been passed down in specific families and some are more prevalent in individuals of certain ethnic descent. (10)

For example, carriers of certain mutations of the BRCA1 or the BRCA2 gene (especially Ashkenazi Jewish women) are at a higher risk of both breast cancer and ovarian cancer, often at an earlier age than the general population. (11) Individuals of Ashkenazi Jewish descent are also at increased risk for inheriting Tay-Sachs, Gaucher, Canavan disease, and Familial Dysautonomia--genetic diseases that can occur when both parents have an abnormal gene that can cause the disease in the child. (10) Testing for diseases such as cystic fibrosis, Fragile X syndrome, Marfan's syndrome, and a host of other diseases are also available today.

What is on the molecular diagnostics horizon?

The 21st century is barely underway, and molecular diagnostics is the hottest topic in the clinical laboratory field. Making molecular diagnostics widely available means overcoming obstacles and resolving issues that have surfaced in its development. In the next segment of "A brief history of medical diagnosis and the birth of the clinical laboratory," Part 5b will detail the most up-to-date testing methods and pertinent automation technologies, and examine the impact these will have on everyday healthcare decisions.

References

1. Berger D. A brief history of medical diagnosis and the birth of the clinical laboratory; Part 4--Fraud and abuse, managed care and lab consolidation. Medical Laboratory Observer. December 1999;

2. Human genome sequence completed. http://cnn.health.printthis.clickability.com/pt/cpt?action+cpt&title=CNN.com+-+Human+g... Accessed August 24, 2006.

3. Kaufman HW, Strom CM. From peapods to laboratory medicine: molecular diagnostics of inheritable diseases. Medical Laboratory Observer. July 2003;30. on peapods

4. Auyang SY. Scientific convergence in the birth of molecular biology. 7. http://www.creatingtechnology.org/biomed/dna.pdf. Accessed August 8,2006.

5. Wikipedia contributors. Gregor Mendel. Wikipedia, The Free Encyclopedia. August 28, 2006, 05:09 UTC. Available at: http://en.wikipedia.org/w/index.php?title=Gregor_Mendel&oldid=72331071. Accessed August 28, 2006.

6. Wikipedia contributors. Thomas Hunt Morgan. Wikipedia, The Free Encyclopedia. August 1, 2006, 03:28 UTC. Available at: http://en.wikipedia.org/w/index.php?title=Thomas_Hunt_Morgan&oldid=66975748. Accessed August 28, 2006.

7. Patrinos GP, Ansorg W. Molecular Diagnostics: Past, Present, and Future. Elsevier Inc. 2005;1, 2-4.

8. A Science Odyssey: People and Discoveries: Watson and Crick describe structure of DNA 1953. http://www.pbs.org/wgbh/aso/databank/entries/do53dn.html. Accessed August 8, 2006.

9. Ardell D. Rosalind Franklin. http://www.accessexcellence.org/RC/Ab/BC/Rosalind_Franklin.html. Accessed August 25, 2006

10. Wright R. TIME 100: James Watson and Francis Crick. http://www.time.com/tim/time100/scientist/profile/watsoncrick.html. Accessed August 8,2006.

11. Genetic Testing for Inherited Diseases. http://www.labtestsonline.org/understanding/conditions/pregnancy-12/html.

12. Fergus K, Simonsen J. The Testing Process in the Ashkenzai Jewish Population. http://www.genetichealth.com/BROV_Genetic_Testing_in_People_of_Ashke-nazi_Jewish_Descent.shtml.

CE test on THE FOUNDATION OF MOLECULAR SCIENCE AND GENETICS

MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in offering continuing education units (CEUs) for this issue's article on THE FOUNDATION OF MOLECULAR SCIENCE AND GENETICS. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E.[R] program (Provider No. 0001) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article on page 10, answer the following test questions and send your completed test form to NIU along with the nominal fee of $20. Readers who pass the test successfully (scoring 70% or higher) will receive a certificate for 1 contact hour of P.A.C.E.[R] credit. Participants should allow four to six weeks for receipt of certificates.

The fee for this continuing education test is $20.

All feature articles published in MLO are peer-reviewed.

Learning Objectives and CE test questions were prepared by Sharon M. Miller, Professor Emeritus, College of Health and Human Sciences, Northern Illinois University, DeKalb, IL.

1. An 1865 publication by an Augustinian monk reported experimental data on which he based his theory of the inheritance of certain traits in pea plants. The researcher was

a. Charles Darwin.

b. A.R. Wallace.

c. Gregor Mendel.

d. Erich von Tschermak.

2. Beginning of modern genetics is traced to the independent discovery and verification of this earlier work by three researchers, early in the 20th century, including

a. Hugo de Vries.

b. Nettie Stevens.

c. Erwin Chargaff.

d. Hermann J. Muller.

3. The proposal of "inborn errors of metabolism," that certain human diseases are the result of altered enzyme activity arising from "defective" hereditary information, was put forward in 1908 by

a. George Beadle.

b. Oswald Avery.

c. Archibald Garrod.

d. Frederick Griffith.

4. The discovery that the particles or genes carried on chromosomes are the mechanical basis of heredity was made by

a. Barbara S. Ross.

b. Nancy Wexler.

c. Colin MacLeod.

d. Thomas H. Morgan.

5. Designation as a "molecular disease" was first applied by what scientist to the disorder arising from a change in a single amino acid residue in the beta chain of the human Hbg molecule?

a. Linus Pauling.

b. Barbara McClintock.

c. Louise Brown.

d. Boyd Morrison.

6. Using advanced X-ray diffraction techniques, what scientist discovered crucial keys to DNA's structure?

a. Rosalind Franklin.

b. Annie Chang.

c. Gobind Khorana.

d. Lap-Chee Tsui.

7. A description of the molecular structure of DNA as a double helix with matching, interlocking base pairs stabilizing the molecule is based on a 1953 article published by

a. Pauling and Franklin.

b. Watson and Crick.

c. Cavendish and Chargaff.

d. Delbruck and Wilkins.

8. Who isolated and purified DNA polymerase 1, the first enzyme that could be used to synthesize DNA in vitro, and was co-recipient of the 1959 Nobel Laureate in Medicine/Physiology?

a. Fred Sanger.

b. Maclyn McCarty.

c. Arthur Kornberg.

d. Frank Stahl.

9. The genetic code was "cracked" when Marshall Nirenberg, Heinrich Mathaei, and Severo Ochoa demonstrated that a sequence of ______ nucleotide bases (a codon) determines each of 20 amino acids.

a. three

b. four

c. five

d. six

10. The 1975 International Meeting at Asilomar Conference Center in Pacific Grove, CA, urged the adoption of guidelines regulating

a. animal and human cloning.

b. prenatal diagnosis of inherited diseases.

c. Stem-cell harvesting.

d. recombinant DNA research.

11. Invention of what molecular biology technique made possible easy amplification of DNA and thereby clinical laboratory provision of such tests as detection of genetic diseases, DNA forensic identification, paternity establishment, analysis of ancient DNA, and the diagnosis of infectious diseases?

a. PCR.

b. FISH.

c. Southern Blotting.

d. Northern Blotting.

12. The first human hormone produced utilizing recombinant DNA technology was

a. somatostatin.

b. insulin.

c. thyroxin.

d. calcitonin.

13. The first company founded to use human genetic information to "discover, develop, manufacture, and commercialize novel medicines for serious and life-threatening diseases" was

a. Chiron.

b. Genentech.

c. Amgen.

d. Aventis.

14. Discovery of the first human cancer-causing gene or oncogene was announced by three research teams working independently in what year?

a. 1971.

b. 1981.

c. 1991.

d. 2001.

15. Testing for increased risk of what inherited disorder is especially critical among individuals of Ashkenazi Jewish descent--one whose ancestry can be traced to the Jews of Central and Eastern Europe?

a. Tay-Sachs disease.

b. Gaucher disease.

c. Canavan disease.

d. All of the above.

16. The first human gene sequenced coded for a transmembrane protein, which, if altered, causes

a. hereditary hemochromatosis.

b. cystic fibrosis.

c. Huntington's disease.

d. Fragile X syndrome.

17. Who proved that genes code for proteins?

a. Tatum and Beadle.

b. Sutton and Temin.

c. Hershey and Chase.

d. Hardy and Weinberg.

18. Officially launched in 1990 and coordinated by the U.S. Department of Energy and the National Institutes of Health, the Human Genome Project was a cooperative international achievement by geneticists to

a. identify the approximately 30,000 genes in human DNA.

b. determine the sequences of the 3 billion chemical base pairs comprising human DNA.

c. develop faster, more efficient methods for DNA sequencing and sequence analysis and transfer these technologies to industry.

d. study the ethical, legal, and social implications of project findings.

e. All of the above.

19. A majority of states have passed genetic non-discrimination bills largely in response to concerns raised as part of the Human Genome Project.

a. TRUE

b. FALSE

20. Although technically feasible, gene replacement therapy has never been attempted in humans.

a. TRUE

b. FALSE

21. Challenges for widespread application of molecular diagnostics include all of the following EXCEPT

a. providing affordable testing and adequate genetic counseling services.

b. convincing payers of the benefits of providing payment coverage.

c. developing adequate proficiency testing and consistency among laboratories.

d. withholding test results because findings might upset the patient.

[GRAPHIC OMITTED]

By Carren Bersch, Editor

Parts 1-4 in this series--originally published from July through December 1999--were written by Darlene Berger, MLO editor from 1998-2000.

Figure 1. History of genetics timeline

1858   Charles Darwin, Alfred Russel Wallace
       Joint announcement of the theory of natural selection: that
       members of a population who are better adapted to the environment
       survive and pass on their traits.
1859   Charles Darwin
       Published The Origin of Species.
1866   Gregor Mendel
       Published the results of his investigations of the inheritance of
       "factors" in pea plants.
1900   Carl Correns, Hugo de Vries, Erich von Tschermak
       Mendel's principles were independently discovered and verified,
       marking the beginning of modern genetics.
1902   Walter Sutton
       Pointed out the inter-relationships between cytology and
       Mendelism, closing the gap between cell morphology and heredity.
1905   Nettie Stevens, Edmund Wilson Independently described the
       behavior of sex chromosomes: XX determines female; XY determines
       male.
1908   Archibald Garrod
       Proposed that some human diseases are due to "inborn errors of
       metabolism" that result from the lack of a specific enzyme.
1910   Thomas Hunt Morgan
       Proposed a theory of sex-linked inheritance for the first
       mutation discovered in the fruit fly, Drosophila, white eye. This
       was followed by the gene theory, including the principle of
       linkage.
1927   Hermann J. Muller
       Used x-rays to cause artificial gene mutations in Drosophila.
1928   Fred Griffith
       Proposed that some unknown "principle" had transformed the
       harmless R strain of Diplococcusto the virulent S strain.
1931   Harriet B. Creighton, Barbara McClintock
       Demonstrated the cytological proof for crossing-over in maize.
1941   George Beadle, Edward Tatum Irradiated the red bread mold,
       Neurospora, and proved that the gene produces its effect by
       regulating particular enzymes.
1944   Oswald Avery, Colin MacLeod, Maclyn McCarty
       Reported that they had purified the transforming principle in
       Griffith's experiment and that it was DNA.
1945   Max Delbruck
       Organized a phage course at Cold Spring Harbor Laboratory which
       was taught for 26 consecutive years. This course was the training
       ground of the first two generations of molecular biologists.
late
1940s  Barbara McClintock
       Developed the hypothesis of transposable elements to explain
       color variations in corn.
1950   Erwin Chargaff
       Discovered a one-to-one ratio of adenine to thymine and guanine
       to cytosine in DNA samples from a variety of organisms.
1951   Rosalind Franklinin
       Obtained sharp X-ray diffraction photographs of DNA.
1952   Martha Chase, Alfred Hershey
       Used phages in which the protein was labeled with 35S and the DNA
       with 32P for the final proof that DNA is the molecule of
       heredity.
1953   Francis Crick
       James Watson
       Solved the three-dimensional structure of the DNA molecule.
1958   Matthew Meselson, Frank Stahl
       Used isotopes of nitrogen to prove the semiconservative
       replication of DNA.
1958   Arthur Kornberg
       Purified DNA polymerase I from E coli, the first enzyme that made
       DNA in a test tube.
1966   Marshall Nirenberg, H. Gobind Khorana
       Led teams that cracked the genetic code: that triplet mRNA codons
       specify each of the twenty amino acids.
1970   Hamilton Smith, Kent Wilcox Isolated the first restriction
       enzyme, Hindll, that could cut
       DNA molecules within specific recognition sites.
1972   Paul Berg, Herb Boyer
       Produced the first recombinant DNA molecules.
1973   Joseph Sambrook
       Led the team at Cold Spring Harbor Laboratory that refined DNA
       electrophoresis by using agarose gel and staining with ethidium
       bromide.
1973   Annie Chang, Stanley Cohen
       Showed that a recombinant DNA molecule can be maintained and
       replicated in E coli.
1975   International meeting at Asilomar, California urged the adoption
       of guidelines regulating recombinant DNA experimentation.
1977   Fred Sanger
       Developed the chain termination (dideoxy) method for sequencing
       DNA.
1977   The first genetic engineering company (Genentech) is founded,
       using recombinant DNA methods to make medically important drugs.
1978   Somatostatin became the first human hormone produced using
       recombinant DNA technology.
1981   Three independent research teams announced the discovery of human
       oncogenes (cancer genes).
1983   James Gusella
       Used blood samples collected by Nancy Wexler and her coworkers to
       demonstrate that the Huntington's disease gene is on chromosome
       4.
1985   Kary B. Mullis
       Published a paper describing the polymerase chain reaction (PCR),
       the most sensitive assay for DNA yet devised.
1988   The Human Genome Project began with the goal of determining the
       entire sequence of DNA composing human chromosomes.
1989   Alec Jeffreys
       Coined the term DNA fingerprinting and was the first to use DNA
       polymorphisms in paternity, immigration, and murder cases.
1989   Francis Collins, Lap-Chee Tsui Identified the gene coding for the
       cystic fibrosis transmembrane conductance regulator protein
       (CFTR) on chromosome 7 that, when mutant, causes cystic fibrosis.
1990   First gene replacement therapy: T cells of a four-year old girl
       were exposed outside of her body to retroviruses containing an
       RNA copy of a normal ADA gene. This allowed her immune system to
       begin functioning.
1993   FlavrSavr tomatoes, genetically engineered for longer shelf life,
       were marketed.

This adapted chart was developed by Jo Ann Lane, a 1994 Woodrow Wilson
National Fellowship Foundation's National Leadership Program for
Teachers participant, and is used through the courtesy of WWNFF
Leadership Program for Teachers (www.woodrow.org/lpt/LPTnational.php)
and Access Excellence @ the National Health Museum
(www.accessexcellence.org/AE/AEPC/WWC/1994/geneticstln. html).

Figure 2. Molecular diagnostics

1949  Characterization of sickle-cell anemia as a molecular disease
1953  Discovery of the DNA double helix
1958  Isolation of DNA polymerases
1960  First hybridization techniques
1969  In situ hybridization
1970  Discovery of restriction enzymes and reverse transcriptase
1975  Southern blotting
1977  DNA sequencing
1983  First synthesis of oligonucleotides
1985  Restriction fragment length polymorphism analysis
1985  Invention of PCR
1986  Development of flourescent in situ hybridization (FISH)
1988  Discovery of the thermostable DNA polymerase: Optimization of PCR
1992  Conception of real time PCR
1993  Discovery of structure-specific endonucleases for cleavage assays
1996  First application of DNA microarrays
2001  First draft versions of the human genome sequence
2001  Application of protein profiling in human diseases

The timeline of the principal discoveries in the field of molecular
biology, which influenced the development of molecular diagnostics.
This adapted chart originally appeared in Molecular Diagnostics: Past,
Present, and Future by George P. Patrinos and Wilhelm Ansorge.
Permission to reprint has been requested from Corporate Relations,
Elsevier.

Figure 3. Challenges for widespread application of molecular diagnostics

* Defining the appropriate circumstances for ordering
* Developing therapies that correct or address specific genetic defects
  or genetic risk factors
* Developing consensus on standards of care
* Educating physicians and patients concerning the potential information
  from testing
* Providing access to affordable testing and services
* Providing adequate controls to prevent discrimination at work, within
  communities and for insurance coverage
* Performing tests on platform technologies that are easy to control
* Developing adequate proficiency testing and consistency among
  laboratories
* Training a sufficient number of medical technologists to perform
  highly complex testing
* Interpreting the results in the context of the clinical history and
  other results
* Integrating results with other family members and ethnicity, while
  complying with HIPAA
* Providing adequate genetic counseling support for physicians and
  patients
* Obtaining adequate reimbursement for performing the tests and the
  potential liability
* Obtaining necessary regulatory approval and coding for reimbursement
* Convincing carriers and payers of the merits of providing payment
  coverage

Challenges for widespread application of molecular diagnostics.
Originally printed in MLO July 2003, "From peapods to laboratory
medicine: molecular diagnostics of inheritable diseases, p. 30.