Perkin Elmer 9600 & 9700 PCR

So much has changed in the last half-decade in the world of gene amplification.

New breakthroughs and protocols have exceeded scientists' wildest expectations for applications of this essential technology. Is there a single life science lab in the country without a thermal cycler? From the high-end, ultrafast light-based cyclers to the tiny personal cyclers, there is a cycler to fit every need.

Way back in the early years of PCR, "doing an amplification" meant simply that--increasing the concentration of some target sequence of interest to use in insertion/
ligation reactions. Quickly enough, scientists developed new ways to use the cycler. Now PCR-based methods exist for every aspect of biology that relies on genetic information.

Forensic, genomic, and evolutionary studies have benefited greatly from the technique known as random amplified polymorphic DNA (RAPD) PCR, which is used to fingerprint species from genomic DNA without prior sequence knowledge. Using arbitrary oligos, two cycles of PCR with low stringency are followed by formal high-stringency PCR with specific primers. The reaction is then followed by RFLP or comparative analysis.

Touchdown PCR was developed to increase the yield of specific products. In this method, the annealing temperature is lowered one degree every second cycle to a lower limit or "touchdown" temperature. This touchdown temperature is then used for 10 cycles. This process ostensibly enriches for a specific product by taking advantage of differences in melting temperatures between correct and incorrect products by twofold per cycle. More recently, this technique has been adapted for genomic sequencing to elucidate DNA sequences from known peptide sequences.

Long PCR is a method that allows amplification of long stretches of DNA, from 0.5 to 20 kb. An enzyme mix containing both proofreading and polymerase activities allows specific extension over great distances. When used as an alternative to RFLP analysis, it shortens the process from six to eight weeks down to hours or days. It also may be used as an alternative to variable number of tandem repeats (VNTR) analysis.

Chromosomal analysis has also benefited from PCR. DOP-PCR is a method that allows uniform amplification of sequences across a genome. Comparative DOP-PCR is used to detect chromosomal imbalances in genetic diseases and evolutionary studies. Amplified fragment length polymorphism-PCR (AFLP-PCR) is used to detect allelic loss in aging and other studies. Single nucleotide polymorphism (SNP) detection has made great strides with PCR. For hybrid cell studies researchers have developed Alu-PCR to detect human chromosomes in the hybrid cells.

Scientists involved in the Human Genome Project have made great use of the cycle sequencing method. Developed as an alternative to the Sanger method, PCR-based sequencing uses femtomolar amounts of primers to start the elongation PCR, followed by sequencing. A major advantage is that this method can be used for linear double-stranded DNA, unlike the Sanger method. It can also be used for cosmid, plaque, and colony DNA sequencing.

Scientists studying RNA expression have not been left out of the PCR craze. Using reverse transcriptase to produce cDNA gave rise to RT-PCR. This method has been used to diagnose a variety of infectious and genetic diseases.

Histologists quickly realized the advantage of having precise control over temperature and incubation time and adapted the cycler for use with in situ hybridization. One limitation of traditional ISH is detection of low-copy number RNAs. This was overcome by the development of cycling-primed in situ (cycling PRINS) labeling, which allowed researchers to gain deeper insight into the more ephemeral processes of the cell.

PE Biosystems*** holds the exclusive license rights to the PCR technology. Anyone who wishes to perform automated PCR using certain reagents and machines must first obtain a license. Fortunately, many reagents and machines include these rights in the purchase price, but the user must check with the supplier before performing the amplification. This applies to research as well as applied fields. If the license is not supplied, PE Biosystems will provide one for a fee.

So what the heck is 'real time' PCR ?

Real-time PCR assays used for quantitative RT-PCR combine the best attributes of both relative and competitive (end-point) RT-PCR in that they are accurate, precise, capable of high throughput, and relatively easy to perform.

To truly appreciate the benefits of this technology, a review of PCR fundamentals is necessary. At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. Exactly when the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made.

For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase.

Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically. As more labs and core facilities acquire the instrumentation required for real-time analysis, this technique may become the dominant RT-PCR-based quantitation technique.

Real-time Reporters: SYBR® Green, TaqMan®, and Molecular Beacons

All real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, the signal of which increases in direct proportion to the amount of PCR product in a reaction. In the simplest and most economical format, that reporter is the double-strand DNA-specific dye SYBR® Green (Molecular Probes). SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases.

The advantages of SYBR Green are that it's inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles.

The two most popular alternatives to SYBR Green are TaqMan® and molecular beacons, both of which are hybridization probes relying on fluorescence resonance energy transfer (FRET) for quantitation.

TaqMan Probes are oligonucleotides that contain a fluorescent dye, typically on the 5' base, and a quenching dye, typically located on the 3' base. When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing, resulting in a nonfluorescent substrate. TaqMan probes are designed to hybridize to an internal region of a PCR product. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5' exonuclease activity of the polymerase cleaves the probe. This separates the fluorescent and quenching dyes and FRET no longer occurs. Fluorescence increases in each cycle, proportional to the rate of probe cleavage.

Molecular beacons also contain fluorescent and quenching dyes, but FRET only occurs when the quenching dye is directly adjacent to the fluorescent dye. Molecular beacons are designed to adopt a hairpin structure while free in solution, bringing the fluorescent dye and quencher in close proximity. When a molecular beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Unlike TaqMan probes, molecular beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement.

Real-time Reporters for Multiplex PCR

TaqMan probes and molecular beacons allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products.

Taqman vs. SYBR Green: The Data

An example of the precision and accuracy of real-time RT-PCR is shown in Figure 1. Panels A and B (left) are the amplification profiles of serial 107-fold dilutions of a cDNA synthesis reaction (107 fold range), amplified with Ambion's QuantumRNA™ 18S Universal primers and detected by either SYBR Green (panel A) or TaqMan (panel B) chemistries. Standard curves for both are shown on the right. The only practical difference between the performance of these two methods is the occasional presence of a false signal late in the SYBR Green amplification. This usually corresponds to a signal lower than that expected from femtograms of RNA, and is of little consequence to accuracy except if a target RNA is extremely rare.

Real-time PCR requires an instrumentation platform that consists of a thermal cycler, computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data. Real-time PCR machines are not cheap, currently about $60-$95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis.

You can count on GMI to provide real time thermal cycling instrumetation

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