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What Are Restriction Site-Associated DNA (RAD) Markers
Introduction
Restriction sites are the unique sites on the DNA that are recognized by restriction enzymes and then cleaved into smaller fragments. They are also called recognition sites. These restriction sites and their recognition has revolutionized the field of genetic engineering ad cloning techniques. Several molecular markers are developed based on this.
In this series restriction site-associated DNA (RAD) markers have been developed in recent years that have been used in the determination of single nucleotide polymorphisms and the construction of the genetic map. Every site pertaining to a particular restriction enzyme can be represented genome-wide by these short RAD tags.
Restriction Site-Associated DNA (RAD) Tags
RAD tags are obtained from the RAD locus and are generally pieces of DNA that have been amplified by a particular method to produce a RAD library. This library contains many RAD tags in it. These tags are used for quantitative trait loci analysis (QTL) and SNP analysis.
RAD markers were first identified in D. melanogaster or fruit fly by Eric Johnson and William Cresko in 2006 by DNA microarray and were later used as a next-generation marker.
Separation of RAD Tags
One of the most important aspects of RAD tags is that it uses the DNA sequences that are flanking the restriction site and its density depends on the type of restriction enzyme used during the process.
There are other methods like AFLP and RFLP which use restriction sites for the detection of the genetic polymorphism RAD uses a reduced-representation method for the same.
The first step in this procedure involves the digestion of double-stranded DNA using a suitable restriction endonuclease to produce the flanking sequences.
In the next step a biotinylated adapter is added to these flanking sequences. Biotinylating is the process of adding biotin to the protein molecule. Biotin has a very high affinity for avidin and streptavidin and can be used as probes.
Once the segments are biotinylated, they are subjected to shear forces that cause the contraction fragments, these fragments are then isolated using streptavidin beads and are used for DNA microarray analysis.
But the procedure discussed above is error-prone, so to reduce the error rate and make it a high throughput method instead of biotinylating the molecules, Illumina platforms are used.
This procedure involves the digestion of DNA molecules using a suitable restriction enzyme and then ligating it with a P1 adapter (first adapter).
After ligation it is subjected to shear forces to contract the DNA fragments which are then ligated to the P2 adapter (second adapter).
When both the adapters are ligated, the fragment is subjected to PCR amplification to specifically amplify the fragment that contains both adapters.
The P1 adapter has a molecular identifier which is nothing but a small DNA sequence barcode that identifies the DNA samples that have been sequenced and pooled in the same reaction.
Identification of RAD Markers
After the separation of the RAD markers, they can be used for the detection of single nucleotide polymorphism. Restriction site-associated markers are these polymorphic sites on the DNA.
Earlier hybridization of RAD tags to microarrays was used for the detection of RAD markers, but because of the lower sensitivity of microarrays, there are chances that it can disrupt the restriction site and subsequent loss of RAD tags. To avoid this situation high throughput sequencing technologies have been used nowadays.
Also, the density of genetic markers obtained is much lower than high throughput technologies.
Modification in RAD Tag Method
ddRAD seq
Here dd stands for double digest RAD sequencing method. This technique was developed in 2012 to reduce the cost conventional RAD tag sequencing method. In this method, two restriction enzymes were used instead of one and the shearing step used for the contraction of DNA is replaced.
This method can be used for whole genome sequencing, to study population differentiation and its adaptation to a given condition.
hyRAD Method
This method was developed in 2016 as an extension of the ddRAD method. This method is very powerful as it helps in the sequencing of orthologous loci even when the DNA is degraded.
In this method enrichment of shotgun libraries is done for the fragment of interest after they are generated by ddRAD seq and their use as a hybridization capture probe.
This method does not depend on the presence of restriction sites, and it has better coverage of loci in the DNA.
Applications of RAD Markers
RAD markers due to their cost effectiveness, and high throughput in studying the quantitative trait loci, evolutionary genetics, population study, association mapping, and ecological genetics.
It is one of the popular reduced-representation library sequencing methods which decreases the complex nature of the genome which can lead to better coverage of fragments flanking the restriction sites.
Conclusion
The restriction site-associated technique is a powerful tool for genome sequencing and has several advantages over traditional restriction-associated techniques like RFLP, AFLP, and RAPD by the fact that it can detect, verify and score markers at the same time. It can be used even for those organisms whose reference genomes are not available.
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