The Human Genome Project - Laying The Groundwork For Thesequencing Of The Human Genome

During the 1960s and 1970s the techniques that would enable the study of molecular genetics were developed. In 1964 the American virologist Howard Temin (1934–94) worked with ribonucleic acid (RNA) viruses and discovered that Crick's central tenet—that DNA makes RNA, and RNA makes protein—did not always hold true. In 1965 Temin described the process of reverse transcriptase—that genetic information in the form of RNA could be copied into DNA. The enzyme called reverse transcriptase used RNA as a template for the synthesis of a complementary DNA strand. Throughout the 1960s American biochemists Robert William Holley (1922–93), Har Gobind Khorana (1922–), and Marshall Warren Nirenberg (1927–), along with American geneticist Philip Leder (1934–), all contributed to deciphering the genetic code by determining the DNA sequence for each of the twenty most common amino acids. Holley, Khorana, and Nirenberg were awarded the 1968 Nobel Prize in physiology or medicine.

American biochemist Paul Berg (1926–) created the first recombinant DNA in 1972, and his work paved the way for isolating and cloning genes. Recombinant DNA is formed by combining segments of DNA, frequently from different organisms. In 1975 the British molecular biologist E. M. Southern (1938–) developed a method to isolate and analyze fragments of DNA that remains in use today. Known as the Southern blot analysis, it is a technique for separating DNA fragments by electrophoresis (a technique that separates molecules based on their size and charge) and identifying a specific fragment using a DNA probe. It is used in genetic research, forensic (related to legal proceedings) examinations of DNA evidence, and clinical medical practice. In 1977 English biochemist Frederick Sanger (1918–), whose many accomplishments have been acknowledged by two Nobel Prizes, and his colleagues developed techniques to determine the nucleic acid base sequence for long sections of DNA. In 1978 Hamilton O. Smith (1931–), Werner Arber (1929–), and Daniel Nathans (1928–99) were awarded the Nobel Prize for an array of discoveries made during the FIGURE 7.1
Single nucleotide polymorphism
SOURCE: Perry Cregan, "Single Nucleotide Polymorphism," in Soybean Genomics and Improvement Laboratory, USDA Beltsville Agricultural Research Center, July, 2003, http://bldg6.arsusda.gov/~pooley/soy/cregan/snp.html (accessed February 20, 2005) 1960s, including the use of restriction enzymes, which ignited the biotechnology field. Restriction enzymes recognize and cut specific DNA sequences. The same year restriction fragment length polymorphisms (DNA sequence variants) were discovered. Figure 7.1 shows a single nucleotide polymorphism—single base changes between homologous DNA fragments.

Using these new genetic techniques, several genes for serious human disorders were identified during the 1980s. In 1982 American molecular biologist James Gusella (1952–)and his colleagues at Harvard University began studying patients with Huntington's disease and determined that the gene for this degenerative, neuropsychiatric disorder was located on the short arm of chromosome 4. During the same year, a gene for neurofibromatosis type I was found on the long arm of chromosome 17. Neurofibromatoses are a group of genetic disorders that cause tumors to grow along various types of nerves and can affect the development of nonnervous tissues such as bones and skin. The disorder may also result in developmental abnormalities such as learning disabilities.

In 1983 American biochemist Kary Banks Mullis (1944–) and his colleagues at the Cetus Corporation in California pioneered the polymerase chain reaction, a fast, inexpensive technique that amplified small fragments of DNA to make sufficient quantities available for DNA sequence analysis—that is, determining the exact order of the base pairs in a segment of DNA. Since it enabled researchers to make an unlimited number of copies of any piece of DNA, it was dubbed "molecular photocopying," and in 1993 Mullis was awarded the Nobel Prize for this tremendous breakthrough in gene analysis. By 1987 automated sequencers were developed, enabling even more rapid sequencing and analysis on large segments of DNA. Figure 7.2 shows the steps involved in a polymerase chain reaction.

FIGURE 7.2
Steps in a polymerase chain reaction (PCR)
SOURCE: "Figure 3. Polymerase Chain Reaction (PCR)," in "Genetic Analysis in the Laboratory," Genetic Biodiversity, The National Biological Information Infrastructure, Center for Biological Informatics of the U.S. Geological Survey, http://genetics.nbii.gov/basic2.html (accessed February 21, 2005)

In 1985 Chinese-Canadian molecular geneticist Lap-Chee Tsui (1950–) and his research team mapped the gene responsible for cystic fibrosis, the most common inherited fatal disease of children and young adults in the FIGURE 7.3
Cytogenic map of human chromosomes
SOURCE: "Cytogenic Map," in Talking Glossary of Genetic Terms, U.S. Department of Health and Human Services, National Institutes of Health, National Human Genome Reseach Institute, http://www.genome.gov/Pages/Hyperion//DIR/VIP/Glossary/Illustration/cytogenetic_map.shtml (accessed February 21, 2005) United States, to the long arm of chromosome 7. The gene for cystic fibrosis was discovered in 1989, and it was determined that three missing nucleic acid bases occurred in the altered gene of 70% of patients with cystic fibrosis.

The mutations associated with Duchenne muscular dystrophy were identified in 1987. This gene is located close to the gene for chronic granulomatous disease (an X-linked autosomal recessive disorder that, if left untreated, is fatal in childhood) on the short arm of the X chromosome. In 1991 Mary-Claire King (1946–) found the first evidence that a gene on chromosome 17 (now known as BRCA1) could potentially be associated with an inherited predisposition to breast and ovarian cancer.

The discoveries and technological advances made by researchers during the 1970s and 1980s gave rise to modern clinical molecular genetics. The study of chromosome structure and function, called cytogenetics, produced methods to view distinct bands on each chromosome. Figure 7.3 is a cytogenetic map of human chromosomes. Cytogenetic studies are applied in three broad areas of medicine: congenital (from birth) disorders, prenatal diagnosis, and neoplastic diseases (cancer).