Representational difference analysis

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Representational Difference Analysis (RDA) is a technique used in biological research to find sequence differences in two genomic or cDNA samples. Genomes or cDNA sequences from two samples (i.e. cancer sample and a normal sample) are PCR amplified and differences analyzed using subtractive DNA hybridization. This technology has been further enhanced through the development of representation oligonucleotide microarray analysis (ROMA), which uses array technology to perform such analyses. This method may also be adapted to detect DNA methylation differences, as seen in Methylation-Sensitive Representational Difference Analysis (MS-RDA)[1].

Theory[edit]

This method relies on PCR to differentially amplify non-homologous DNA regions between digested fragments of two nearly identical DNA species, that are called 'driver' and 'tester' DNA. Typically, tester DNA contains a sequence of interest that is non-homologous to driver DNA. When the two species are mixed, the driver sequence is added in excess to tester. During PCR, double stranded fragments first denature at ~95°C and then re-anneal when subjected to the annealing temperature. Since driver and tester sequences are nearly identical, the excess of driver DNA fragments will anneal to homologous DNA fragments from the tester species. This blocks PCR amplification and there is no increase in homologous fragments. However, fragments that are different between the two species will not anneal to a complementary counterpart and will be amplified by PCR. As more cycles of RDA are performed, the pool of unique sequence fragment copies will grow faster than fragments found in both species.

Technical aspects of how amplification occurs using universal adapters is described below in greater detail.

Procedure[edit]

Amplify mRNA representations (optional)[edit]

This is used when there is only a small amount of starting material (mRNA) available.

You start with two tubes. One with "Tester" mRNA (i.e. from a cancer sample) and the other with "Driver" mRNA (i.e. from a normal sample). cDNA is synthesized from these samples using a technique such as random priming. The newly formed cDNA in the two tubes are cut with a restriction endonuclease such as DpnII. The cDNA (possessing sticky ends) can then be ligated to an adapter "R". With a known adapter attached to the cDNAs, PCR can be used to amplify the samples. Again, both samples in both tubes can be cut with the same restriction enzyme (DpnII) to remove adapter "R".

Denaturation and Subtractive Hybridization[edit]

Now you have plenty of "Tester" cDNA (i.e. from cancer samples) and "Driver" cDNA (i.e. from normal samples). To the "Tester" cDNA, an adapter, "J", is added. The "Tester" cDNA and the "Driver" cDNA are mixed (it is helpful to add excess Driver cDNA). The mixed sample with both Tester and Driver cDNA can be heated to about 95C to denature the strands. Then, the temperature can be held at around 55C for hybridization. This creates a few possible outcomes, "Tester" cDNA bound to "Tester" cDNA, "Tester" cDNA bound to "Driver" cDNA, and "Driver" cDNA bound to "Driver" cDNA.

PCR amplification to enrich "Tester" cDNA bound to "Tester" cDNA[edit]

After the first round of denaturation and hybridization, it is important to fill-in the ends of the fragments. Thus, "Tester" cDNA bound to itself will now have adapter "J" at each end on each strand. You can then run PCR with primers that can recognize a sequence on adapter "J". Thus, "Tester" cDNA bound to itself will be exponentially enriched. "Tester" cDNA bound to "Driver" cDNA will only be linearlly enriched.

Repeat subtractive hybridization and PCR amplification to further enrich "Tester" cDNA bound to "Tester" cDNA[edit]

Following PCR, you can digest the single stranded PCR products with mungbean nuclease. Using restriction enzyme DpnII will remove adapter "J", and another adapter, "N", can be added to the "Tester" cDNA bound to "Tester" cDNA PCR products. Now, the sample can undergo another round of denaturation and hybridization, and PCR amplification. This further enriches the "Tester" cDNA bound to "Tester" cDNA, which are the genes that are differentially expressed in "Tester" mRNA (e.g. only found in cancer samples and not normal samples).

References[edit]

  • Lisitsyn N, Lisitsyn N, Wigler M. (1993), Cloning the differences between two complex genomes. Science, 259, 946-951

External links[edit]