The polymerase chain reaction (PCR), a cell-free method for amplifying DNA, was first developed in the mid-1980s. It was both very fast and readily allowed parallel amplifications of DNA sequences from multiple starting DNA samples. If you wanted to amplify each exon of the β-globin gene from blood DNA samples from 100 different individuals with β-thalassemia, a single person could now do that in a very short time. Because of its simplicity, rapidity, and versatility, PCR has revolutionized genetics. PCR uses reaction cycles consisting of consecutive steps that require different temperatures, but alternate methods use constant temperatures; we describe isothermal amplification approaches to amplifying DNA in vitro at the end of this section.
The polymerase chain reaction (PCR): basic features and quantitation
For most purposes, PCR is used for selective amplification. It relies on using a heat- stable DNA polymerase to synthesize copies of a small, pre-determined DNA segment of interest within a complex starting DNA (such as total genomic DNA from easily accessed blood or skin cells). To initiate the synthesis of a new DNA strand, a DNA polymerase needs a single-stranded oligonucleotide primer that is designed to bind to a specific complementary sequence within the starting DNA.
For the primer to bind preferentially at just one desired location in a complex genome, the oligonucleotide often needs to be about 20 nucleotides long or more and is designed to be able to base-pair perfectly to its intended target sequence (the strength of binding depends on the number of base pairs formed and the degree of base matching).
To allow the primer to bind, the DNA needs to be heated. At a high-enough temperature, the hydrogen bonds holding complementary DNA strands together are broken, causing the DNA to become single-stranded. Subsequent cooling allows the oligonucleotide primer to bind to its perfect complementary sequence in the DNA sample (annealing or hybridization). Once bound, the primer can be used by a suitably heat-stable DNA polymerase for it to synthesize a complementary DNA strand.
In PCR, two primers are designed to bind to complementary target sequences that are close to each other on the same DNA molecule but on opposing DNA strands. The primer binding sites are chosen to flank the region of interest, and the primers are normally designed to be convergent so that each new strand is synthesized in the direction toward the sequence bound by the other primer. In further cycles of DNA denaturation, primer binding, and DNA synthesis, previously synthesized DNA strands become targets for binding by the other primer, causing a chain reaction (Figure 1).
Fig1. The polymerase chain reaction (PCR). The reaction usually consists of about 30 cycles of (1) DNA denaturation, (2) binding of oligonucleotide primers flanking the desired sequence, and (3) new DNA synthesis in which the desired DNA sequence is copied and primers are incorporated into the newly synthesized DNA strands. Numbers in the vertical strips to the left indicate the origin of the DNA strands, with original DNA strands represented by 0 and PCR products by 1 (made during the first cycle), 2 (second cycle), or 3 (third cycle). The first cycle will result in new types of DNA product with a fixed 5′ end (determined by the primer) and variable 3′ ends (extending past the other primer). After the second cycle, there will be two more products with variable 3′ ends but also two desired products of fixed length (shown at left by filled red squares) with both 5′ and 3′ ends defined by the primer sequences. Whereas the products with variable 3′ ends increase arithmetically (amount = 2n where n is the number of cycles), the desired products initially increase exponentially (amount = 2n−1) until the reaction reaches a stationary phase as the amount of reactants becomes depleted (see Figure 2). After 25 or so cycles, the desired product accounts for the vast majority of the DNA strands.
The end result is that millions of DNA copies (amplicons) can be made of just the desired DNA sequence of interest within the complex starting DNA. By amplifying the desired sequence, we can now study it in different ways—by directly sequencing the amplified DNA, for example. PCR can also be used to analyze RNA transcripts. In that case the RNA transcripts are first converted into cDNA using reverse transcriptase (the process is called reverse transcription-PCR or RT-PCR).
Quantitative and real-time PCR
In routine PCR, all that is required is to generate a detectable or usable amount of product. But for some purposes, there is a need to quantify the amount of product. There are different types of quantitative PCR. Some are variants of routine PCR and use standard PCR machines to give a relative quantification of a sequence of interest within test samples and controls.
Real-time PCR is a form of quantitative PCR carried out in specialized PCR machines. It provides both absolute quantification (the absolute number of copies) and also relative quantification. Instead of waiting for the end of the reaction, the measurements are performed within the PCR machine while the reaction is still progressing. That is, the amplified DNA is tracked by detecting and quantifying in real time the fluorescence from a reporter molecule included within the reaction mixture. Fluorescently-labeled PCR products from the exponential phase of the reaction (see Figure 2 for the different phases of a polymerase chain reaction) are removed and analyzed to measure the ratio of the fluorescence exhibited by the PCR product from a test sample (for example, one that is associated with disease or that is suspected of being abnormal) to the fluorescence exhibited by the PCR product from a control sample. The basis of the quantitation is that during the exponential phase, the amount of PCR product is proportional to the amount of target DNA sequence in the input DNA. We expand on this method when we consider important applications in profiling gene expression, and in later chapters we also describe its use in assays for altered nucleotides in DNA.
Fig2. Different phases in a polymerase chain reaction (PCR). After a lag phase, the amount of PCR product increases gradually at first. In the exponential phase, beginning after about 16–18 cycles and continuing to approximately the 25th cycle, the amount of PCR product is taken to be proportional to the amount of input DNA; quantitative PCR measurements are made on this basis. With further cycles, the amount of product increases at first but then tails off as the saturation phase approaches, when the reaction efficiency diminishes as reaction products increasingly compete with the remaining primer molecules for template DNA.
Advantages and disadvantages of PCR
Because of its simplicity and speed PCR is widely used for myriad research purposes and diagnostic applications. It is also exquisitely sensitive and robust. Thus, it can success fully amplify DNA fragments from tiny amounts of tissue samples that may have been badly degraded, and even from single cells. As a result, there have been numerous applications in forensic and archaeological studies, and in research and diagnostic applications that involve single cells. PCR is robust enough to allow analysis of tissue samples that have been fixed in formalin.
Because PCR involves reaction cycles with steps at very high temperatures, prokaryotic heat-stable DNA polymerases are used (isolated from thermophilic bacteria or archaea that naturally live in hot springs or hydrothermal vents in oceans), and they can have comparatively high error rates. Taq DNA polymerase isolated from the bacterium Thermus aquaticus is popularly used, but it lacks a 3′-to-5′ exonuclease proof reading function and so has a significant base-misincorporation rate (about 1 in 105). More recently, heat-stable DNA polymerases with 3′-to-5′ exonuclease activity and lower error rates have become popular, such as ones isolated from archaea belonging to the Pyrococcus genus (including Pfu DNA polymerase and the VentTM and Deep VentTM DNA polymerases).
PCR has two major disadvantages compared to cell-based DNA cloning. First, unlike cell-based DNA amplification, scaling up to give very large quantities of amplified DNA is not practical. Second, PCR is unsuited to amplifying large DNA sequences: the vast majority of applications produce amplicons less than 10 kb in length (whereas sequences up to a few hundred kilobases in length can be cloned in bacteria and DNA sequences longer than 1 Mb can be cloned in yeast cells).
Isothermal amplification is an alternative to PCR for amplifying DNA sequences in vitro
PCR is not the only type of method to allow amplification of DNA in vitro. As the name indicates, isothermal amplification means that the in vitro DNA amplification is carried out at a constant temperature. Isothermal amplification has the advantage that there is no need for specialized equipment to carry out complicated thermocycling; a simple water bath at a constant temperature will suffice. The isothermal amplification methods are, however, not so versatile as PCR, and are primarily used as diagnostic and detection techniques rather than for DNA cloning purposes.
Most of the isothermal methods are designed to allow amplification of specific sequences (and some examples, including the popular LAMP method, are listed in Table 1). In addition, some isothermal amplification methods allow indiscriminate amplification of all sequences in a complex starting nucleic acid, as described below.
Table1. EXAMPLES OF SOME ISOTHERMAL AMPLIFICATION METHODS PERMITTING EXPONENTIAL AMPLIFICATION OF SPECIFIC SEQUENCES
Nonselective DNA amplification methods to provide sufficient material for study from samples with limited starting nucleic acid
Up until now we have considered PCR as a tool for selective amplification of a desired DNA sequence so that it can be purified, then studied or put to use in some way, or as a way of detecting a DNA sequence in some clinical or other diagnostic test. But both PCR and isothermal amplification methods can also permit indiscriminate amplification of all DNA fragments from a starting DNA. The object is simply to increase the amount of DNA for study.
This type of application is especially required when the source of DNA is in very limiting quantities, such as genomic DNA retrieved from bone samples at archaeological and historical sites, or from tiny human residues left at crime scenes, or when analyzing single cells, whether in pre-implantation diagnosis or for research purposes. it has also become essential for preparing DNA libraries for many high throughput (“next-generation sequencing”) methods where millions of different DNA fragments in the starting DNA need to be amplified (in order to increase the signals from incorporating individual nucleotides so that they can be detected during the sequencing reaction).
Various types of PCR can be used to amplify all sequences in a complex starting population. One popular approach is to attach a common DNA sequence onto the ends of all the fragments, and then use primers that are specific for the common flanking sequence. The first step is to prepare a double-stranded adaptor oligonucleotide (sometimes also called a linker oligonucleotide) by designing complementary synthetic oligonucleotides and allowing them to hybridize. The common adaptor oligonucleotide is ligated to all of the DNA fragments. Then, primers specific for the universal adaptor sequence are used to amplify any fragments that have the adaptor sequence at their ends (Figure 3).
Fig3. Nonselective DNA amplification using double-stranded adaptor oligonucleotides. Here the idea is to ligate a universal sequence to the ends of a heterogeneous collection of DNA fragments so that all DNA fragments can be amplified using a single set of primers. Two synthetic oligonucleotides are designed to have complementary sequences and allowed to hybridize to form the universal sequence. After the resulting double stranded adaptor oligonucleotide is ligated to the DNA fragments, amplification of all of the DNA fragments is possible using primers that are specific for the adaptor sequence.
For some applications the nonselective amplification method requires that the amplified DNA fragments be flanked by two different adaptor sequences. For example, in high-throughput DNA sequencing methods that require amplified DNA templates, DNA fragments to be sequenced are often manipulated so that different adaptor sequences are bolted on at the two ends.
Isothermal amplification methods have also been applied to allow nondiscriminate amplification. Whole-genome amplification is possible from single cells using, for example, the multiple displacement amplification (MDA) method. It relies on using the highly processive bacteriophage Φ29 DNA polymerase that can produce amplicons greater than 70 kb in length. Because of its importance in whole-genome amplification from single cells.
