Frequently asked questions:
Last updated September 1, 2006
The only equipment necessary is a real-time PCR machine. While these machines are expensive to purchase (ours cost $22,000), most researchers should have access to one at their institution. The machine must allow you the flexibility to program repeated fluorescence measurements while the samples are held at the annealing temperature. Standard one-color detection machines are fine (any machine that can detect FAM can detect SYBR). A gradient feature is useful, because it can be used to automate the denaturation process, but it is not necessary--the denaturation could be done by hand, or the denatured samples and references could be run on two separate machines or at separate times on the same machine.
The most expensive part of the assay is the cost of the nucleic acid preparation and the initial PCR reaction. The only additional costs are the additional time spent using the real time PCR machine, and the costs of exonuclease I and SYBR Green dye (about a dollar per sample).
I was able to teach a research assistant in our lab how to perform the assay in several days, using the protocol provided on this website. The steps of an AmpliCot experiment will be straightforward for anyone with basic molecular biology experience. The biggest hurdle is setting up an organized method to track samples, their annealing data, and their analyses.
AmpliCot is cheaper than cloning and sequencing--even high throughput sequencing. The costs of sequencing can be prohibitive if many highly diverse samples must be studied. In many cases the sequences themselves are not informative, and AmpliCot provides an economical measurement of sequence diversity.
We designed AmpliCot using off-the-shelf reagents and machines. Machines built specifically to perform annealing kinetics measurements might provide more precise measurements in a shorter amount of time, and might increase the automation of the process. The thermal degradation of SYBR Green is a major source of error in our measurements, and a more thermostable dye, or another method for measuring duplex formation (such as FRET or UV absorbance) could potentially improve the assay.
Many methods, such as Immunoscope/Spectratyping, heteroduplex analysis, and Vbeta antibody staining, are able to detect expanded clones in the repertoire, but until now, the only way to quantify the number of receptors in the unexpanded repertoire was to make extrapolations from exhaustive sequencing experiments. AmpliCot offers a quantitative measurement of diversity rapidly and at a low cost.
The major cost is in aquiring the sample, purifying it (e.g. through flow sorting) and extracting the nucleic acids. The cost for the remainder of the assay is little more than that of a PCR reaction.
There are several important issues to consider up front. First, are you interested in a general diversity measurement, or are you specifically interested in the diversity of a specific subset of lymphocytes (e.g. naive CD4+ cells)? You may well have to sort the samples before performing the assay. Second, the diversity assay is a property of the sample as a whole, and cannot be normalized for differences in RNA yield or integrity, or differences in cell number or purity. This means that you must be very careful in how you process samples, and perform routine quality control after sorts and RNA preps. Third, the immune repertoire is very large, and this necessarily affects the sample sizes one must use in the assay. Ideally, to fully sample the repertoire, one must sample at least three times the number of cells in a single copy of the repertoire. This means samples of many millions of cells for naive repertoire measurements. Such large samples are often difficult to obtain.
We have used two sorts of standards: known numbers of cloned T cell receptors, and known numbers of very diverse lymphocytes (e.g. pooled naive cells from several donors).
We have used AmpliCot to look at TCR beta chain diversity in mice and humans. There is no reason the assay could not be adapted to look at other TCR chains, immunoglobulin genes, or other species, as long as the relevant sequence information is known.
I see two applications as particularly promising. First, AmpliCot might be used for molecular ecology projects that now rely on exhaustive sequencing of PCR products. Using AmpliCot would make it possible to measure the diversity of many more samples, making new and larger experimental designs feasible. Second, many experiments, from DNA computing to combinatorial chemistry to in vitro evolution all make use of complex DNA libraries which undergo repeated selection procedures. AmpliCot could potentially be used to measure the diversity of these libraries before and after each selection step.
The major considerations are (1) is knowing the actual sequence data very important (2) do I expect enough samples and enough complexity to make AmpliCot more cost effective than simply sequencing, and (3) are the sequences I am studying suitable for AmpliCot. To be suitable, all sequences (or a representative subset) must be amplifiable with closely spaced primers so that stringency conditions can be found to distinguish between homoduplexes and heteroduplexes.