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. 1, 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.

Fig1. Schematic diagram of polymerase chain reaction. Repetitive cycles of denaturation, annealing, and extension are paced by  temperature cycling of the reaction. Two primers indicated as short segments anneal to opposite template strands (long line) to define the region  to be amplified. Extension occurs from the 3′ ends (half-arrowheads). In each cycle, genomic DNA is denatured and annealed to primers that  extend in opposite directions across the same region, producing long products of undefined length. Long products generated by extension of one  of the primers anneal to the other primer during the next cycle, producing short products of defined length. Any short products present also produce more short products. After n cycles, up to 2n new copies of the amplified region are present—n long products and (2nn − n) short products plus one original genomic copy. A similar approach can be used to amplify RNA targets by initial reverse transcription of the RNA template to  produce the DNA template. (From Burtis CA, Ashwood ER, Bruns DB: Tietz fundamentals of clinical chemistry, ed 6, St Louis, 2008, Saunders.)

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. 1, 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

 Reverse Transcriptase Polymerase Chain Reaction

If the nucleic acid of interest is RNA rather than DNA, the PCR procedures can be modified to include the conversion of RNA to DNA using reverse transcriptase (RT) in the initial steps. RT-PCR is useful in the identification of RNA viral agents, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV).

Multiplex Polymerase Chain Reaction

Multiplex PCR uses numerous primers in a single reaction tube to amplify nucleic acid fragments from different targets. Specific nucleic acid amplification should occur if the appropriate target DNA is present in the sample tests. Detection may be accomplished by the traditional Southern transfer method and subsequent nucleic acid probe, by enzyme immunoassay (EIA) methods, or by gene chip analysis. This technology is limited by the following: (1) the number of primers that can be included in a single reaction; (2) primer-primer interference; and (3) nonspecific nucleic acid amplification.

Real-Time Polymerase Chain Reaction

Real-time PCR uses fluorescence-resonance energy transfer to quantitate specific DNA sequences of interest and identify point mutations. Real-time PCR is particularly appealing because the procedure is less susceptible to amplicon contamination and is more accurate in quantifying the initial copy number.

Other Amplification Techniques

Strand Displacement Amplification Strand displacement amplification (SDA) is a fully automated method that amplifies target nucleic acid without the use of a thermocycler. A double-stranded DNA fragment is created and becomes the target for exponential amplification.

Transcription-Mediated Amplification

Transcription-mediated amplification (TMA) is another iso thermal assay that targets DNA or RNA, but generates RNA as its amplified product. TMA is used to detect microorganisms (e.g., Mycobacterium tuberculosis).

Nucleic Acid Sequence–Based Amplification

 Nucleic acid sequence–based amplification (NASBA) is simi lar to TMA, but only RNA is targeted for amplification. Its applications include the detection and quantitation of HIV and detection of cytomegalovirus (CMV).

Ligase Chain Reaction Nucleic Acid Amplification

 Oligonucleotide pairs hybridize to target sequences within the gene or the cryptic plasmid. The bound oligonucleotides are separated by a small gap at the target site. The enzyme DNA polymerase uses nucleotides in the ligase chain reaction (LCR)–nucleic acid amplification reaction mixture to fill in this gap, creating a ligatable junction. Once the gap is filled, DNA ligase joins the oligonucleotide pairs to form a short, single-stranded product that is complementary to the original target sequence. This product can itself serve as a target for hybridization and ligation of a second pair of oligonucleotides present in the LCR reaction mixture.

Subsequent rounds of denaturation and ligation lead to the geometric accumulation of amplification product. The amplified products can be detected in an LCx analyzer (Abbott Laboratories, Abbott Park, Ill) by microparticle EIA.

Target Enrichment Strategies

 In the last 10 years, next generation sequencing (NGS) technologies have been developed. This approach overcomes the limitations of traditional Sanger sequencing by providing highly parallel sequencing with a separate sequence result for every sequence of interest. This has positioned NGS as the method of choice for targeted re-sequencing of regions of the human genome. Two main technologies have been used to enable target enrichment: PCR and hybridization.

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مواضيع ذات صلة

Characteristics of Nucleic Acids

solation of Integration-Defective Mutants

Gene Order Permutation and the Campbell Model

Simultaneous Deletion of Chromosomal and Lambda DNA

Mapping Integrated Lambda

Expression Patterns of Developmental Genes

Enhancer Traps for Detecting and Cloning Developmental Genes

Cloning Developmental Genes

General Considerations on Signaling

DNA Strand Inheritance and Switching in Fission Yeast

DNA Cleavage at the MAT Locus

Glucose Metabolism