Molecular Studies

Clinical laboratory and surgical pathology testing has become more restricted in recent years, often as a result of limitations imposed by third-party insurers. However, some of the restrictions are not for unwelcome financial considerations alone but rather for welcome increased diagnostic specificities from new technologies that turn from broad, nonspecific screening algorithms to disease- and diagnosis-specific tests. The most technologically advanced procedures are related to direct analysis of the genetic material contained within neoplasms. Direct analysis of a cell's genetic constitution is the most specific method for arriving at a pathologic diagnosis. The PCR technique is fundamental to this new methodology.

FIGURE 12.14. Immunohistochemical algorithm for identifying spindle cell sarcomas.

FIGURE 12.15. Graphic depiction of forward scatter and side scatter. (A) The smaller cellular elements of blood (lymphocytes) are clustered to the lower portion of the scattergram (less forward scatter) and to the left side (less side scatter). (B) The larger and more complex cells (monocytes and granulocytes) will cluster higher up and also move toward the right (increasing forward and side scatter). (Courtesy of Dr. David Lawrence, MD|

FIGURE 12.15. Graphic depiction of forward scatter and side scatter. (A) The smaller cellular elements of blood (lymphocytes) are clustered to the lower portion of the scattergram (less forward scatter) and to the left side (less side scatter). (B) The larger and more complex cells (monocytes and granulocytes) will cluster higher up and also move toward the right (increasing forward and side scatter). (Courtesy of Dr. David Lawrence, MD|

It was Kornberg and Ochoa's work on DNA replication that allowed the development of all the individual research and clinical components that are the basis of all molecular studies. For this monumental contribution, these biochemists were awarded the 1959 Nobel Prize in physiology/medicine.71

PCR allows the generation of several million copies of a specific region or segment of DNA or RNA. Analysis of these copies then permits specific diagnoses related to specific changes in the genome of the analyzed cell. Developed in 1987 by Mullis and Faloona,72,73 PCR methods use template (double-stranded) DNA, two oligonucleotide primers (20-30 mers) that are base complements of the 3' ends of the region of interest on the template DNA, a mixture of the four deoxynucleotides (dATP, dCTP, dGTP, dTTP), appropriate buffers, ions, and

Taq polymerase, a thermally stable enzyme isolated from the bacterium Thermus aquaticus. The enzyme is critical in DNA replication of the bacterium and is simply a variant of the same enzyme characterized by Kornberg and Ochoa. The enzyme was isolated in the 1960s by bacteriologist Thomas Brock from a bacterium isolated from a hot spring in Yellowstone National Park.74

Once the genetic segment has been amplified, it must be analyzed for its specific content. The easiest and most commonly used method is simple gel elec-trophoresis followed by ethidium bromide staining and visualization under ultraviolet light. An example of this gel separation is shown in Figure 12.17, which also demonstrates the sequential reactions taking place in the amplification cycles. The variation in the distance traveled by the PCR fragments depends on the amount

FIGURE 12.16. DNA gene/ nucleotide sequencing chromatogram. This technique allows for individual nucleic acid identification and any changes from a known sequence— deletion, addition, or substitution. (Courtesy of Dr. Timothy Formosa, MD)

FIGURE 12.17. Electrophoretic gel separation of PCR-generated DNA sequences. (A) Sequence fragments that have the same nucleic acid sequence will migrate the same distance through the gel. (B) Even a single nucleic acid variation (addition, substitution, or deletion) will cause the PCR fragment to assume a different spatial configuration, resulting in a change in how far the segment will travel through the gel. (Courtesy of Dr. O. Henegariu, New Haven, CT.)

FIGURE 12.17. Electrophoretic gel separation of PCR-generated DNA sequences. (A) Sequence fragments that have the same nucleic acid sequence will migrate the same distance through the gel. (B) Even a single nucleic acid variation (addition, substitution, or deletion) will cause the PCR fragment to assume a different spatial configuration, resulting in a change in how far the segment will travel through the gel. (Courtesy of Dr. O. Henegariu, New Haven, CT.)

of the deoxynucleotides incorporated into the fragments during the actual amplification process.75

0 0

Post a comment