Restriction site associated DNA markers
Encyclopedia
Restriction site Associated DNA (RAD) markers are a type of genetic marker
that can be used for genetic mapping. The use of RAD markers for genetic mapping is often called RAD mapping. An important aspect of RAD markers and mapping is the process of isolating RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction enzyme
site throughout the genome. Once RAD tags have been isolated, they can be analyzed to identify and/or genotype DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs). Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers.
The initial procedure to isolate RAD tags involved digesting DNA with a particular restriction enzyme, ligating biotinylated adapters to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, and isolating the biotinylated fragments using streptavidin beads. This procedure was used to isolate RAD tags for microarray analysis.
More recently, the RAD tag isolation procedure has been modified for use with high-throughput sequencing on the Illumina platform. The new procedure involves digesting DNA with a particular restriction enzyme, ligating the first adapter to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, preparing the sheared ends and ligating the second adapter, and using PCR to specifically amplify fragments that contain both adapters. Importantly, the first adapter contains a short DNA sequence barcode and different DNA samples can be prepared with different barcodes to allow for sample tracking when multiple samples are sequenced in the same reaction. The use of high-throughput sequencing to analyze RAD tags has been termed RAD tag sequencing, RAD sequencing, RAD-Seq, or RADSeq.
, and the use of high-throughput sequencing to analyze RAD tags has been termed RAD tag sequencing, RAD sequencing, RAD-Seq, or RADSeq.
Prior to the development of high-throughput sequencing technologies, RAD markers were identified by hybridizing RAD tags to microarrays. Due to the low sensitivity of many microarrays, this approach can only detect either DNA sequence polymorphisms that disrupt restriction sites and lead to the absence of RAD tags or substantial DNA sequence polymorphisms that disrupt RAD tag hybridization. Therefore, the genetic marker density that can be achieved with microarrays is much lower than what is possible with sequencing.
Genetic marker
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species. It can be described as a variation that can be observed...
that can be used for genetic mapping. The use of RAD markers for genetic mapping is often called RAD mapping. An important aspect of RAD markers and mapping is the process of isolating RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
site throughout the genome. Once RAD tags have been isolated, they can be analyzed to identify and/or genotype DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs). Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers.
Isolation of RAD tags
RAD tags are the DNA sequences that immediately flank each instance of a particular restriction enzyme site throughout a genome and the process of isolating RAD tags is an important aspect of RAD markers and mapping. Different RAD tag densities can be achieved by using different restriction enzymes during the isolation process.The initial procedure to isolate RAD tags involved digesting DNA with a particular restriction enzyme, ligating biotinylated adapters to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, and isolating the biotinylated fragments using streptavidin beads. This procedure was used to isolate RAD tags for microarray analysis.
More recently, the RAD tag isolation procedure has been modified for use with high-throughput sequencing on the Illumina platform. The new procedure involves digesting DNA with a particular restriction enzyme, ligating the first adapter to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, preparing the sheared ends and ligating the second adapter, and using PCR to specifically amplify fragments that contain both adapters. Importantly, the first adapter contains a short DNA sequence barcode and different DNA samples can be prepared with different barcodes to allow for sample tracking when multiple samples are sequenced in the same reaction. The use of high-throughput sequencing to analyze RAD tags has been termed RAD tag sequencing, RAD sequencing, RAD-Seq, or RADSeq.
Detection and genotyping of RAD markers
Once RAD tags have been isolated, they can be analyzed to identify and/or genotype DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs). DNA sequence polymorphisms that are identified and genotyped by isolating RAD tags are referred to as RAD markers. The most efficient way to analyze RAD tags is by high-throughput DNA sequencingDNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
, and the use of high-throughput sequencing to analyze RAD tags has been termed RAD tag sequencing, RAD sequencing, RAD-Seq, or RADSeq.
Prior to the development of high-throughput sequencing technologies, RAD markers were identified by hybridizing RAD tags to microarrays. Due to the low sensitivity of many microarrays, this approach can only detect either DNA sequence polymorphisms that disrupt restriction sites and lead to the absence of RAD tags or substantial DNA sequence polymorphisms that disrupt RAD tag hybridization. Therefore, the genetic marker density that can be achieved with microarrays is much lower than what is possible with sequencing.