Polymerase chain reaction

A common molecular biology technique is the PCR method, which amplifies a single strand of nucleic acid by several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence (Fig 3-3). This method relies on thermal cycles of repeated heating and cooling of the DNA sample for thermal denaturation (DNA melting) and enzymatic replication. The components required for selective and repeated amplification are primers, which are short DNA fragments that contain sequences complementary to the target region (cDNA), DNA polymerase, and nucleotides. The selectivity of PCR is due to the use of these primers. Successful DNA extraction from various tissues and fluids with PCR techniques is dependent on the condition of the specimen.

Table 3-3 Summary of Molecular Techniques Used in Diagnostic Pathology

PCR techniques have advanced considerably in recent years, and there are now approximately 20 PCR variants. The clinical relevance of detecting a PCR product depends on numerous variables, including the primers selected, laboratory controls, and demographic considerations. Thus, for clinicians making a clinicopathologic diagnosis, PCR is best used as an adjunct to routine pathologic diagnostic techniques. See also Part III, Genetics, in BCSC Section 2, Fundamentals and Principles of Ophthalmology.

Microarray

Scientists and clinicians use microarrays to survey the expression of thousands of genes in a single assay, the output of which is called a gene expression profile (GEP). Microarray technology can be used to further our understanding of fundamental aspects of human growth and development, explore the molecular mechanisms underlying normal and dysfunctional biological processes, and elucidate the genetic causes of many human diseases. Different types of microarrays are available, including DNA microarrays (the most common type), microRNA microarrays (MMChips), protein microarrays, tissue microarrays (Fig 3-4), cellular (or transfection) microarrays, antibody microarrays, and carbohydrate (glycoarray) microarrays.

Figure 3-3 Polymerase chain reaction (PCR). A, PCR starts with a denaturing step in which DNA samples are heated to 95°C to separate the target double-stranded DNA into single strands. B, The temperature is then lowered to 55°C to allow the primers to anneal to their complementary sequences. The primers are designed to bracket the DNA region to be amplified. C, The temperature is raised to 72°C to allow polymerase to attach at each priming site and extend or synthesize a new DNA strand between primer sequences, producing 2 new DNA molecules. D, Step C is repeated numerous times to generate thousands to millions of copies.

(Courtesy of Theresa R. Kramer, MD.)

The basic process underlying all of the DNA microarray platforms is straightforward: a glass slide or chip is spotted or “arrayed” with oligonucleotides or DNA fragments (called probes) that represent specific gene-coding regions. Fluorescently or chemiluminescently labeled purified cDNA or cRNA (called target) is hybridized to the arrayed slide or chip. After the chip is washed, the raw data are obtained by laser scanning, entered into a database, and analyzed with statistical methods.

Although DNA microarrays were initially developed to quantify the expression of a limited number of genes of clinical relevance, the technology has also been applied to tumor diagnosis and drug resistance in malignancies. Validating the results of microarray experiments is a critical step in the analysis of gene expression. Quantitative (real-time) PCR is the preferred method for validating gene expression profiling.

Figure 3-4 Tissue microarrays are constructed with small core biopsies of different tumors/ tissues. A core is trephined from the donor paraffin block of the tumor (a). A recipient paraffin block is prepared, creating empty cores (b). The cores are incorporated into the slots (c) until all are occupied (d). Glass slides are prepared and stained from the paraffin block containing the cores with a selected antibody (e). Microscopic examination reveals the different staining patterns of each core (f).

(Courtesy of Patricia Chévez-Barrios, MD.)

Clinical use of PCR and microarray

Routine clinical use of PCR and microarray was traditionally limited to the diagnosis of leukemias, lymphomas, soft-tissue neoplasms, and tumors with nondiagnostic histopathology results. These procedures are now increasingly used in the detection of infectious agents (eg, the herpesvirus family), in tumor prognostication (eg, uveal melanoma), and in detection of genetic alterations that are amenable to targeted therapies (eg, cutaneous melanoma and hematologic malignancies). Some current commercial microarray and PCR platforms can be used to stratify biopsy-sized tumor samples based on the metastatic potential of the tumor.

The selection of commercially available microarray and PCR kits continues to grow. The ongoing refinement and wider commercial availability of molecular genetic techniques will likely lead to wider integration of these modalities into clinical practice and the pathologic evaluation of biopsy specimens. However, the cost of these testing modalities is often significantly higher than that of other, more traditional, diagnostic modalities and should be discussed with patients before tests are ordered. See Part III, Genetics, in BCSC Section 2, Fundamentals and Principles of Ophthalmology, for further discussion of molecular genetics.

• Khong JJ, Moore S, Prabhakaran VC, Selva D. Genetic testing in orbital tumors. Orbit. 2009; 28(2–3):88–97.

• Liu L, Li Y, Li S, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol. 2012;2012:251364.

• Vaziri K, Schwartz SG, Kishor K, Flynn HW Jr. Endophthalmitis: state of the art. Clin Ophthalmol. 2015;9:95–108.

Excerpted from BCSC 2020-2021 series: Section 4 - Ophthalmic Pathology and Intraocular Tumors. For more information and to purchase the entire series, please visit https://www.aao.org/bcsc.