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.