DNA and RNA are polymers made up of repeating nucleotides or bases that are linked together (Fig. 14-1). DNA and RNA have the same two purine bases, adenine (A) and guanine (G), but the pyrimidine bases differ. DNA has cytosine (C) and thymine (T); RNA substitutes uracil (U) for T. DNA is predominantly a double-stranded molecule with specific base pairs linked together (Fig. 14-2). Nucleotides are bonded together and two strands are twisted into an alpha helix (Fig. 14-3).

Figure 14-1 A, Purine and pyrimidine bases and the formation of complementary base pairs. *Dashed lines* indicate the formation of hydrogen bonds. B, A single-stranded DNA chain. Repeating nucleotide units are linked by phosphodiester bonds that join the 5′ carbon of one sugar to the 3′carbon of the next. Each nucleotide monomer consists of a sugar moiety, a phosphate residue, and a base. ^∗In RNA, thymine is replaced by uracil, which differs from thymine only in its lack of the methyl group. ^∗∗In RNA, the sugar is ribose, which adds a 2′-hydroxyl to deoxyribose. (Adapted from Piper MA, Unger ER: Nucleic acid probes: a primer for pathologists, Chicago, 1989, ASCP Press.)

Figure 14-2 Structure of DNA.
The DNA molecule is a double helix that consists of two sugar-phosphate backbones with four bases—cytosine *(C),* guanine *(G),* adenine *(A),* and thymine *(T)*—attached. C and G residues and A and T residues on opposite strands pair through hydrogen bonding. (From LeGrys V, Leinbach SS, Silverman L: Clinical applications of DNA probes in the diagnosis of genetic diseases, Crit Rev Clin Lab Sci 25:255, 1987.)

Figure 14-3 The DNA double helix, with sugar-phosphate backbone and pairing of the bases in the core forming planar structures. (From Jorde CB, Carey JC, Bamshead MJ et al, editors: Medical genetics, ed 3, St Louis, 2006, Mosby.)

How Does DNA Replicate?

DNA is a very stable molecule and replication is straightforward. The process of replication (Fig. 14-4) involves one strand of the molecule acting as a template for the creation of a complementary strand. As a result of this process, two identical daughter molecules are produced. In the laboratory, the hydrogen bonds that hold the strands of the double helix can be broken apart or denatured. If complementary strands of DNA are denatured in the laboratory, they can spontaneously rejoin, or anneal. The process of denaturation and annealing (see later discussion) can be used effectively in molecular testing.

Figure 14-4 DNA replication.
Double-stranded DNA is separated at the replication fork. The leading strand is synthesized continuously, whereas the lagging strand is synthesized discontinuously but joined later by DNA ligase. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.)

Production of functional protein from genetically encoded DNA is achieved by two processes, transcription and translation. Transcription is a process of generating a strand of messenger RNA (mRNA) that encodes for the gene and is expressed as a protein. Translation occurs when the mRNA moves from the nucleus of a cell into the cellular cytoplasm to the ribosomes. mRNA is translated into an amino acid sequence on the ribosome. This process manufactures a protein that was originally encoded in DNA in the cellular nucleus.

Forms of RNA

RNA can be easily replicated and is used in molecular laboratory testing. RNA exists in three forms, mRNA, tRNA, and rRNA. All the forms of RNA exist as single-stranded polymers and are longer than DNA. The function of each form of RNA differs, as follows :

• mRNA—translates DNA coding into functional proteins (Fig. 14-5)

Figure 14-5 DNA transcription and mRNA processing.
A gene that encodes for a protein contains a promoter region with a variable number of introns and exons. Transcription commences at the transcription start site. Pre-mRNA is processed by capping, polyadenylation, and intron splicing and becomes a mature mRNA. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.)

• tRNA—transports various amino acids to manufacture proteins

• rRNA—site of protein synthesis directed by mRNA

Amplicons and Amplicon Control Measures

An amplicon is a piece of genetic material, such as DNA, that can be formed as the product of a natural event or artificial amplification technique, such as a polymerase chain reaction (PCR). A molecular diagnostic laboratory that performs in vitro amplification reactions needs to practice techniques to control contamination. This is especially true if a high number of thermal cycles is used for the PCR.

PCR is highly sensitive but a disadvantage to the use of this assay is that it is prone to producing false-positive results. In laboratories in which PCR is performed frequently, any false-positives are generally caused by amplicon contamination. A broken capillary tube or a PCR plate left carelessly at the edge of a table can aerosolize those amplicons, which can then adhere to lab coats and objects in the room.

A simple and effective way to combat amplicon contamination is to wipe down everything—equipment, workstations, and pipettes—with bleach. Generously spray with 10% bleach and then let it sit for 15 to 30 minutes.

Amplification Techniques in Molecular Biology

Polymerase Chain Reaction

The polymerase chain reaction (PCR) is an in vitro method that amplifies low levels of specific DNA sequences in a sample to higher levels suitable for further analysis (Fig. 14-6, A). To use this technology, the target sequence to be amplified must be known. Typically, a target sequence ranges from 100 to 1000 base pairs (bp) in length. Two short DNA primers, typically 16 to 20 bp, are used. The oligonucleotides (small portions of a single DNA strand) act as a template for the new DNA. These primer sequences are complementary to the 3′ ends of the sequence to be amplified.

This enzymatic process is carried out in cycles. Each repeated cycle consists of the following:

• DNA denaturation. Separation of the double DNA strands into two single strands through the use of heat.

• Primer annealing. Recombination of the oligonucleotide primers with the single-stranded original DNA.

• Extension of primed DNA sequence. The enzyme DNA polymerase synthesizes new complementary strands by the extension of primers.

Each cycle theoretically doubles the amount of specific DNA sequence present and results in an exponential accumulation of the DNA fragment being amplified (amplicons). In general, this process is repeated approximately 30 times. At the end of 30 cycles, the reaction mixture should contain about 2³⁰ molecules of the desired product.

After cycling is complete, the amplification products can be analyzed in various ways. Typically, the contents of the reaction vessel are subjected to gel electrophoresis. This allows visualization of the amplified gene segments (e.g., PCR products, bands) and determination of their specificity. Additional product analysis by probe hybridization or direct DNA sequencing is often performed to verify the authenticity of the amplicon further.

Three important applications of PCR are as follows:

1. Amplification of DNA

2. Identification of a target sequence

3. Synthesis of a labeled antisense probe

PCR analysis can lead to the following: (1) detection of gene mutations that signify the early development of cancer; (2) identification of viral DNA associated with specific cancers (e.g., human papillomavirus [HPV], a causative agent in cervical cancer); and (3) detection of genetic mutations associated with various diseases, such as coronary artery disease associated with mutations of the gene that encodes for the low-density lipoprotein receptor (LDLR).

The PCR technique has undergone modifications (see Fig. 14-6, B). One uses nested primers in a two-step amplification process. First, a broad region of the DNA surrounding the sequence of interest is amplified, followed by another round of amplification to amplify the specific gene sequence to be studied. Another PCR modification successfully differentiates alleles of the same gene.

Modified Polymerase Chain Reaction Techniques

Analysis of Amplification Products

Many of the revolutionary changes that have occurred in research in the biological sciences, particularly the Human Genome Project, can be directly attributed to the ability to manipulate DNA in defined ways. Molecular genetic testing focuses on the examination of nucleic acids (DNA or RNA) by special techniques to determine whether a specific nucleotide base sequence is present.

The applications of nucleic acid testing have expanded, despite higher costs associated with testing, in various areas of the clinical laboratory. These include genetic testing, hematopathology diagnosis and monitoring, and identification of infectious agents. Molecular testing has the following advantages:

• Faster turnaround time

• Smaller required sample volumes

• Increased specificity and sensitivity

Conventional Analysis

Detection of DNA products by PCR assay can be conventionally analyzed using agarose gel electrophoresis after ethidium bromide staining. This technique is simply an extra step after a PCR assay has been run. DNA and other biomolecules can be separated based on charge, size, and shape. DNA has a net negative charge and will migrate toward the anode (positive pole). PCR products are loaded into an agarose gel and electrophoresed. Ethidium bromide is a dye that intercalates into nucleic acids and will fluoresce with an orange color under ultraviolet (UV) irradiation. An image analyzer uses UV light to capture computer images of the PCR products.

Other Techniques

Other techniques are used to enhance the sensitivity and specificity of amplification techniques. Probe-based DNA detection systems have the advantage of providing sequence specificity and lower detection limits. Other techniques include the hybridization protection assay, DNA EIA, automated DNA sequencing technology, single-strand conformational polymorphism, and restriction fragment length polymorphism (RFLP) analysis. The selection of one technique over another is often based on factors such as sensitivity and specificity profiles, cost, turnaround time, and local experience.

DNA Sequencing

DNA sequencing is considered to be the gold standard to which other molecular methods are compared. DNA sequencing displays the exact nucleotide or base sequence of a fragment of DNA that is targeted. The Sanger method, which uses a series of enzymatic reactions to produce segments of DNA complementary to the DNA being sequenced, is the most frequently used method for DNA sequencing. Automated sequencing techniques use primers with four different fluorescent labels.

1. The first step in sequencing a target is usually to amplify it by cloning or in vitro amplification, usually PCR. Once the amplified DNA is purified from the clinical specimen (the target DNA), it is heat-denatured to separate the double-stranded DNA (dsDNA) into single strands (ssDNA).

2. The second step involves adding primers to the ssDNA. Primers are short synthetic segments of ssDNA that contain a nucleotide sequence complementary to a short strand of target DNA. The patient’s DNA serves as a template to copy. DNA polymerase catalyzes the addition of the appropriate nucleotides to the preexisting primer. DNA synthesis is terminated when the deoxynucleotide is incorporated into a growing DNA chain.

Branched DNA

Branched DNA (bDNA) is another quantitative test that uses signal amplification instead of target amplification. Target DNA or RNA is hybridized at different sites by two types of probes. Branched DNA assays are used to measure the viral load of hepatitis B virus (HBV), HCV, HIV-1, CMV, and microbial organisms (e.g., Trypanosoma brucei).

The Versant HIV-1 RNA 3.0 assay (bDNA; Bayer, Berkeley, Calif), uses bDNA technology. It is the only viral load assay specifically designed to target multiple sequences of the HIV-1 genome with more than 80 nucleic acid probes.

Hybridization Techniques

Many forms of probe hybridization assays involve the complementary pairing of a probe with a DNA or RNA strand derived from the patient’s specimen. The common feature of probe hybridization assays is the use of a labeled nucleic acid probe to examine a specimen for a specific, homologous DNA or RNA sequence. Clinical probes are usually labeled with nonradioisotopic molecules such as digoxigenin, alkaline phosphatase, biotin, or a fluorescent compound. The detection systems are conjugate-dependent and include chemiluminescent, fluorescent, and calorimetric methodologies.

Liquid-Phase Hybridization

In the liquid-phase hybridization (LPH) assay, the target nucleic acid and labeled probe interact in solution. Specific homologous hybrids are subsequently separated from the remaining nucleic acid component and the hybrids are identified by an appropriate detection system.

Dot Blot and Reverse Dot Blot

These hybridization methods are used in the clinical laboratory for the detection of disorders in which the DNA sequence of the mutated region has been identified (e.g., sickle cell anemia, cystic fibrosis). These techniques are capable of distinguishing the homozygous or heterozygous state of a mutation.

Dot Blot

The dot blot hybridization method detects single-base mutations using allele-specific oligonucleotides (ASOs). Unlike other assays, dot blot does not require enzyme digestion or electrophoretic separation of DNA fragments. The procedure uses labeled oligonucleotide probes of about 15 to 19 bp. DNA is amplified in the region of a known mutation, denatured, and applied to separate areas of a membrane or filter. A probe designed to detect a normal DNA sequence is added to one area; a second probe for the detection of a sequence with the single-base mutation is applied to a second area. Ideally, only the labeled probe whose base sequences perfectly match those of the patient will hybridize.

Reverse Dot Blot

In this variation of the dot blot procedure, the ASO probes are bound to a filter and denatured DNA from the patient is added to the immobilized ASO. Hybridization occurs only if the patient’s DNA contains base sequences that are 100% complementary to those of the probe. A common variation of the reverse dot blot procedure is to bind oligonucleotide probes of a slightly longer length than usual to a 96-well microtiter plate. Biotin is used to label copies of the target sequence. The labeled copies are hybridized in the wells to the bound probes and detected using avidin conjugated to horseradish peroxidase. Subsequent addition of substrate produces a colored reaction that can be read photometrically.

Blotting Protocols

The Southern blot and Northern blot techniques are used to detect DNA and RNA, respectively. These procedures share the following steps:

1. Electrophoretic separation of the patient’s nucleic acid

2. Transfer of nucleic acid fragments to a solid support (e.g., nitrocellulose)

3. Hybridization with a labeled probe of known nucleic acid sequence

4. Autoradiographic or colorimetric detection of the bands created by the probe–nuclei acid hybrid

Southern Blot

Specimen DNA is denatured and treated with restriction enzymes to create DNA fragments; then the ssDNA fragments are separated by electrophoresis (Figure 14-7). The electrophoretically separated fragments are then blotted to a nitrocellulose membrane, retaining their electrophoretic position and hybridized with radiolabeled single-stranded DNA fragments with sequences complementary to those being sought. The resulting dsDNA bearing the radiolabel, if present, is then detected by radiography.

The Southern blot procedure has clinical diagnostic applications for disorders associated with significant changes in DNA, a deletion or insertion of at least 50 to 100 bp (e.g., fragile X syndrome), and determination of clonality in lymphomas of T or B cell origin. If a single-base mutation changes an enzyme restriction site on the DNA, resulting in an altered band or fragment size, the Southern blot procedure can detect these changes in DNA sequences, referred to as RFLPs. Single-base mutations that can be determined by the Southern blot technique include sickle cell anemia and hemophilia A.

Northern Blot

mRNA from the specimen is separated by electrophoresis and blotted to a specially modified paper support; this results in covalent fixing of the mRNA in the electrophoretic positions. Radiolabeled, ssDNA fragments complementary to the specific mRNA being sought are then hybridized to the bound mRNA. If the specific mRNA is present, its radioactivity is detected by autoradiography.

The derivation of this technique from the Southern blot technique used for DNA detection has led to the common usage of the term Northern blot for the detection of specific mRNA. However, the Northern blot technique is not routinely used in clinical molecular diagnostics.

Western Blot

Compared with the Southern blot technique, which separates and identifies RNA fragments and proteins, and the Northern blot technique, which concentrates on isolating mRNA, in the Western blot technique proteins are separated electrophoretically, transferred to membranes, and identified through the use of labeled antibodies specific for the protein of interest (Fig. 14-8).

The Western blot technique detects antibodies to specific epitopes of antigen subspecies. Electrophoresis of antigenic material results in the separation of the antigen components by molecular weight (MW). Blotting the separated antigen to nitrocellulose, retaining the electrophoretic position, and causing it to react with patient specimen will result in the binding of specific antibodies, if present, to each antigenic band. Electrophoresis of known MW standards allows for the determination of the MW of each antigenic band to which antibodies may be produced. These antibodies are then detected using EIA reactions that characterize antibody specificity.

The Western blot technique is often used to confirm the specificity of antibodies detected by enzyme-linked immunosorbent assay (ELISA) screening procedures.

Microarrays

Microarray (DNA chip) technology has helped accelerate genetic analysis, just as microprocessors accelerated computation (Fig. 14-9). Microarrays are basically the product of bonding or direct synthesis of numerous specific DNA probes on a stationary, often silicon-based support. The chip may be tailored to particular disease processes. The technique is easily performed and readily automated.

Figure 14-9 Affymetrix GeneChip probe array. (Courtesy Affymetrix, Santa Clara, Calif.)

Microarrays are miniature gene fragments attached to glass chips. These chips are used to examine the gene activity of thousands or tens of thousands of gene fragments and to identify genetic mutations using a hybridization reaction between the sequences on the microarray and a fluorescent sample. After hybridization, the chips are scanned with high-speed fluorescent detectors and the intensity of each spot is quantitated (Fig. 14-10).

Figure 14-10 Overview of eukaryotic target labeling for GeneChip expression arrays. (Courtesy Affymetrix, Santa Clara, Calif.)

The identity and amount of each sequence are revealed by the location and intensity of fluorescence displayed by each spot. Computers are used to analyze the data (Fig. 14-11).

Figure 14-11 Data from an experiment showing the expression of thousands of genes on a single GeneChip probe array. (Courtesy Affymetrix, Santa Clara, Calif.)

The applications of microarrays in clinical medicine include the analysis of gene expression in malignancies (e.g., mutations in the breast cancer 1 gene [BRCA-1], mutations of the p53 tumor suppressor gene, genetic disease testing, viral resistance mutation detection).

The Human Genome GeneChip set (HG-U133 Set; Affymetrix, Santa Clara, Calif), consisting of two GeneChip arrays, contains almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well-substantiated human genes. The sequence clusters were created from the UniGene database and then refined by analysis and comparison with a number of other publicly available databases (e.g., Washington University EST trace repository and University of California, Santa Cruz, Golden Path human genome database).

The HG-U133A array includes representation of the RefSeq database sequences and probe sets related to sequences previously represented on the Human Genome U95Av2 array. The HG-U133B array contains primarily probe sets representing expressed sequence tag (EST) clusters. The applications of this array include defining tissue and cell type–specific gene expression and investigating cellular and tissue responses to the environment (e.g., heat shock, interactions with other cells, exposure to chemical compounds, growth factors, or other signaling molecules). In addition, this array helps elucidate human cell differentiation by the following: (1) determining which transcripts are increased or decreased during distinct stages in cellular differentiation; and (2) detecting which genes are uniquely expressed during different stages of tumorigenesis.

Another genomic microarray, GenoSensor (Tempe, Ariz), enables researchers to screen for abnormal gene amplifications and deletions with the sensitivity to detect single-gene copy change in a variety of specimens. The GenoSensor system simultaneously screens for gene copy number changes in 287 targets spotted in triplicate. This permits the screening of proto-oncogenes, tumor suppressor genes, microdeletion syndrome, gene regions, and subtelomeric regions.

Next Generation Sequencing Technology

Molecular characterization of tumors typically include Sanger sequencing (described previously) of a limited number of genes known to harbor mutations with well-described clinical appearances. If several genes need to be studied, Sanger sequencing can be costly and time-consuming. Next Generation Sequencing (NGS) technologies have the potential to be more cost-effective and be able to simultaneously sequence complete genomes of patients to deliver personalized medicine. NGS can produce thousands to millions of genome sequences at one compared to the 96 sequences processed by the traditional Sanger method. Currently, Roche, Illumina, and Applied Biosystems manufacture second-generation sequencing platforms.

With NGS technology, the process begins with template preparation by shearing DNA (or cDNA) to create fragment libraries. Adaptor sequences are added to these fragments and serve as primers for amplification usually by emulsion PCR or bridge PCR methods. The resulting amplified signal beads or clusters are analyzed using a variety of platform-specific chemical analyses but all are based on the addition of labeled nucleotides. Digital images are captured and analyzed to determine the sequence of the target DNA. The impact of NGS on cancer treatment is presented in Chapter 33, Tumor Immunology.

Future Directions of Molecular Diagnostic Testing

Nucleic acid testing (NAT) has played an important role in reducing transfused transmitted infections. NAT testing has been adopted in the United States, Canada, France, Australia, New Zealand, and South Africa. Additional countries in Europe and the Far East continue to be added to the list of NAT testing countries. NAT testing includes PCR assays and TMA.

Molecular testing is no longer confined to high-volume reference laboratories. Molecular diagnostics has advanced in precision, accuracy, speed, detection, and cost. Applications range from detection of infectious diseases to cellular and tissue antigens. Molecular diagnostic testing using nucleic acid–based assays provides rapid and accurate diagnosis and identification of infectious diseases that previously involved a long waiting period for pathogen identification. Past challenges, such as contamination, have become less of a problem because of the use of these new techniques.

Increasingly sensitive and specific methods continue to be developed. Molecular techniques are attractive in a wide variety of testing situations and offer promising advancements in laboratory science. New automated systems focus on pathogen detection and multidrug-resistant organisms, particularly health care–acquired (e.g., nosocomial) infections. Developing countries are expanding their use of molecular diagnostics in HIV diagnosis and viral load testing. In cancer detection, however, molecular testing is still considered an immature industry. Genomic testing permits the increased collection of data on large populations in disease research studies.

CASE STUDY

History and Physical Examination

KM, a 38-year-old man, drove himself to the emergency room because of a worsening condition of shortness of breath. He had a sore throat, felt tired, and had a fever, nonproductive cough, and mild chest pain. He had no history of serious medical conditions and was a lifelong nonsmoker.

During the last 5 years, he had been diagnosed with genital herpes and gonorrhea. He reported having persistent diarrhea for the past several months. He also noted a weight loss during the same period. He and his male partner had been having unprotected intercourse for several years. There was no history of IV drug use.

Physical examination revealed an underweight man with palpable lymph nodes. Plaques of Candida albicans were seen in the back of his throat. His chest sounds had diffuse crackles in both lungs.

Laboratory assays and a chest x-ray were ordered. He was also referred to counseling because of his high risk status for HIV-AIDS.

Laboratory Results

Assay	Patient’s Results	Reference Range
Hemoglobin	10.5 g/dL	13.5-16.5 g/dL
Hematocrit	29%	40%-50%
Total leukocyte count	7.0 × 10⁹/L	4.5-10.0 × 10⁹/L
Total lymphocyte count	0.80 × 10⁹/L	1.-3.5 × 10⁹/L
CD4+ T cells	0.04 × 10⁹/L	0.7-1.1 × 10⁹/L
CD8+ T cells	0.41 × 10⁹/L	0.5-0.9 × 10⁹/L
B lymphocytes	0.09 × 10⁹/L	0.2-0.5 × 10⁹/L
ELISA HIV test	Positive	Negative