Representational Difference Analysis
Encyclopedia
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)http://www.ncc.go.jp/en/nccri/divisions/14carc/14carc01_1.html.
Technical aspects of how amplification occurs using universal adapters is described below in greater detail.
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".
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.
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
or cDNA samples. Genomes or cDNA sequences from two samples (i.e. cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
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)
Roma
- Places :Italy* Rome, the capital of Italy, is called Roma in Italian and some other languages* Roma Tre University, a university located in Rome, Italy, and founded in 1992...
, which uses array
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
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)http://www.ncc.go.jp/en/nccri/divisions/14carc/14carc01_1.html.
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 together, 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.
Amplify mRNA representations (optional)
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
DpnII restriction endonuclease family
In molecular biology, the DpnII restriction endonuclease family is a family of restriction endonucleases which includes DpnII from Diplococcus pneumoniae. These enzymes recognise the double-stranded DNA unmethylated sequence GATC and cleave before G-1, where it encompasess the full length of the...
. 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
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 together (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
After the first round of denaturationDenaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
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
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
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.