Representational difference analysis (RDA) is a technique used in biological research to find sequence differences in two
genomic
Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
or
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
samples. Genomes or cDNA sequences from two samples (i.e.
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal bl ...
sample and a normal sample) are
PCR PCR or pcr may refer to:
Science
* Phosphocreatine, a phosphorylated creatine molecule
* Principal component regression, a statistical technique
Medicine
* Polymerase chain reaction
** COVID-19 testing, often performed using the polymerase chain r ...
amplified and differences analyzed using
subtractive DNA hybridization
Subtractive hybridization is a technology that allows for Polymerase chain reaction, PCR-based amplification of only Complementary DNA, cDNA fragments that differ between a control (driver) and experimental transcriptome. cDNA is produced from Mes ...
. This technology has been further enhanced through the development of
representation oligonucleotide microarray analysis (ROMA), which uses
array
An array is a systematic arrangement of similar objects, usually in rows and columns.
Things called an array include:
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Music
* In twelve-tone and serial composition, the presentation of simultaneous twelve-tone sets such that the ...
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).
Theory
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.
References
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*Lisitsyn N, Lisitsyn N, Wigler M. (1993), Cloning the differences between two complex genomes. ''Science'', 259, 946-951
External links
Overview at fullerton.edu
Genomics