Jump to content

User:Phd1684/Molecular Copy-number Counting

From Wikipedia, the free encyclopedia

Molecular Copy-number Counting

Molecular Copy-number Counting (MCC) is a method for determining the abundance of a given sequence of DNA in a sample. By comparing the results for a query sequence with those for a reference sequence, the copy-number (that is, the number of copies per cell) of the query sequence can be accurately determined. The method is accurate and can be applied to extremely small samples, although it cannot be applied simultaneously to the extremely large number of query sequences which can be analysed by some other approaches.

MCC can be used to examine copy-number variation (inherited differences in the number of copies of particular genes or other sequences) and also somatic copy-number changes (the gain or loss of copies of some sequences in some cells, as is commonly observed in cancer). The method is based on the counting of individual molecules of the query sequence.

Many methods exist for measuring copy-number. Most depend on measuring the intensity of a signal (for example, the strength of hybridisation of the sample DNA to a probe on a microarray; or the quantitation of a PCR product from the target sequence). These methods are subject to artefacts which can quantitatively affect the signal strength, and often require considerable quantities of good quality sample DNA (for example, from tens of nanograms to several micrograms). Techniques such as whole-genome amplification can be used as a pre-treatment to increase the amount of DNA from small samples, but these methods run the risk of biasing the subsequent copy-number analysis.

MCC, in contrast, works by amplifying, detecting and thereby counting individual molecules of the query sequence in the sample. At the same time, molecules of a reference sequence (whose copy number in the cells is known) are also counted, for comparison. It is therefore a "digital" approach rather than an "analogue" method, and can give highly accurate results. Moreover, it is inherently suitable for measuring copy numbers in extremely small samples, such as a few tens of cells microdissected from a mixed population in a cancer biopsy, even after fixation, embedding and other histological processes which damage DNA.

The first step in MCC is to dilute the sample to the point where the reference sequence is at a concentration of (typically) around one molecule per ten microlitres. (If the reference sequence is a normal somatic sequence, present at two copies per cell, this dilution is approximately 0.3pg of human DNA per microlitre). This DNA is then dispensed into multiple samples (for example, into the 96 wells of a microtitre plate), so that each sample has a roughly 50% chance of containing a molecule of the reference sequence.

A two-stage PCR is then used to amplify any molecules of the reference sequence and the query sequence present in each sample. In the first stage, primers for both the reference and query sequences are used in a multiplex PCR. These products are then split (for instance, into two replica microtitre plates), and amplified a second time with primers for the reference sequence and the query sequence in two independent (monoplex) reactions. The PCR products from both sets of reactions (reference and query) are then detected, for example by gel electrophoresis or directly by fluorescence.

The numbers of samples scoring positive for the reference sequence, and for the query sequence, are then counted and compared. For example, if the query sequence is present in twice as many samples as the reference sequence, then its copy number must be twice that of the reference. In fact, the analysis is slightly more subtle, using the Poisson distribution to allow for the fact that some poasitive samples may have contained two or more copies of the reference (or query) sequence.

The initial multiplex PCR is conducted at low stringency, and is highly efficient even when many sequences are co-amplified. MCC can therefore be used to measure the copy number of up to several hundred different sequences (including one or more references) simultaneously, although the workload is higher than microarray-based analyses for large numbers of sequences.

To date, MCC has been most used in the analysis of cancer biopsies. The genome of cancer cells often undergoes deletion or multiplication some sequences (for example, loss of genes which inhibit tumour progression; or the gain of extra copies of genes which promote it), and the identification of lost or gained genes can shed light on the mechanism of cancer progression and hence inform both diagnosis and therapy. MCC has proven particularly effective in analysing small numbers of pre-cancerous cells taken from biopsies of lesions at an early stage.

Advantages and drawbacks of MCC

MCC's chief advantage lies in its ability to accurately measure the copy number of multiple sequences in extremely small samples containing poor-quality DNA; and in the relative ease with which new query sequences can be chosen and included in the analysis. However, it is not practical to use it for genome-wide surveys of many thousands of sequences, as can be done using microarrays.






References

[edit]
[edit]