Diversity arrays technology

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Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provides comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. [1] [2] [3] The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), insert fragments into a vector to be placed as probes within a microarray, then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. [1] [2] The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species. [1]

Contents

First reported in 2001 by Damian Jaccoud, Andrzej Kilian, David Feinstein, and Kaiman Peng, DArT prioritized significant advantages over other traditional primer-based methods like the ability to analyze large amounts of various samples from a low amount of initial DNA. [1] [2] [4] [5] It also afforded low costs and faster results compared to related solid state DNA arrays that detected Single Nucleotide Polymorphisms (SNPs). [1] [2] Since its inception, the technology has been a major instrument in the analysis of polyploid plants as well as in the construction of physical and genetic map to understand related on species based on similarities and allelic variances among their genomes. [1] [2] [6] [7] [8] [3]

History

The concept was first developed by Damian Jaccoud, Andrzej Kilian, David Feinstein, and Kaiman Peng in 2001. [1] They aimed to establish a genomic DNA-polymorphism detection and quantification technique that would increase throughput when compared to more traditional methods like Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeats (SSR). [1] [2] [4] [5] They also aimed to minimize cost and reliance on sequenced genomes to identify polymorphisms which is a consequence of early immobilized, solid-states DNA arrays, like DNA chips, which solely identify SNPs. [1] [2] A byproduct of their discovery of a fast, low-cost whole-genome profiling method was that it also provided with the identification of SNPs as well as base-pair insertions, deletions, and shifts, which is an added layer of allelic variation between species analyzed. [1] [2]

Jaccoud, Kilian, Feinstein, and Peng selected nine subspecies of rice as their source for genomic DNA and polymorphism analysis. [1] The analysis consisted of detecting the presence, or absence, of specific DNA polymorphisms with probing arrays as well as quantifying the strength of each signal, via fluorescence, within the subspecies. Upon selecting and extracting DNA samples from subjects, samples were digested with three specific restriction enzymes and ligated with T4 ligase. Following ligation into double stranded DNA, dilution as well as extraction of a short amount of mixture to use as a PCR template was performed. Products were placed into a pCR2.1-TOPO vector and subsequently transformed into E. coli, who were selected based on resistance to ampicillin and pigmentation from the X-gal interaction. [1] [2] Cloned cells are amplified with PCR-amplified, purified, and introduced into a microarray. Reference DNA and samples were mixed with fluorescent dyes, Cy3 or Cy5, mixed, denatured, and allowed to hybridize to further reintroduce them into the microarray for further analysis. Results reported that the use of DArT was able to detect the presence or absence of polymorphism in an expedient manner as compared to RFLP as well as quantify the polymorphisms detected. [1] In addition, DArT was able to minimize the amount of initial DNA required to conduct the analysis significantly compared to other methods. [1]

Procedure

The DArT is broken down into three essential steps: Complexity reduction, genomic representation, and DArT assay. [2]

Complexity reduction

This step of the process deals with reducing large complex genomic DNA of selected species into more, manageable fragmented components through the use of specific restriction enzymes. In addition, this step exclusively relies on digestion enzymes over a couple effort of digestion enzymes and primers due to the reported increased polymorphism identified across analyzed samples. [2] The PstI enzyme is a commonly used restriction enzyme for this step because of its specificity to the nonrepetitive, nonmethylated genome of species. [2] [6] [7] [8] [9]

Genomic representation

Once genomic DNA has been reduced to a manageable size from the previous step by incorporating one or two specific restriction enzymes, the next step involves selecting for the fragments that include largest amount of significant polymorphism across gene pool. These selected fragments are termed “representations” as they are smaller representations of the initial, larger genomic DNA. It is eminent to avoid repetitive sequences when selecting fragments as these will exhibit the lowest amount of polymorphism within analyzed genomic DNA. [2]

DArT assay

Digested sequences are ligated using T4 ligase to produce double stranded DNA. A small amount of ligated mixture will be diluted then amplified via PCR. During PCR, it is important to use primers complementary to the restriction-enzymes’ cutting sites and RedTaq polymerase, which is rarely inhibited. Mix product into an amplified, gene pool representation and ligate onto vector pCR2.1-TOPO. Following representation insertion into vector, transform vector into E. coli cells via electrical shocking or chemical means. Incubate cells and select based on ampicillin resistance and white-pigmentation from inactive β-galactosidase gene in a medium containing X-gal. [1] [2] Inserts are then amplified via PCR and inserted as spotters into a microarray slide. Slides are centrifuge to isolate inserts, which are then purified.

Fluorescent dyes, Cy3 or Cy5, are added to the microarray targets, which are genomic representations. Following addition of the fluorescent dye, targets are added to microarray probes containing the amplified E. coli clones where denaturing and subsequent hybridization, if possible, takes place. Following hybridization, slides are washed and scanned with an imaging system that targets fluorescent signals with the incorporation of an open-source software called DArTsoft. Interactions and dissimilarities between probe and various targets are used to develop a histogram which quantifies and identifies several forms of DNA polymorphism among analyzed genomes. [1] [2]

Applications

Molecular breeding

The ability to identify and quantify allelic variations among genomes without the need for a sequenced genome is of great value to DArT and has large implications in the molecular breeding sector. [1] [2] By comparing crops with phenotypes such as higher yields of produce or resistance to certain environmental parasites, a phenotype can be directly linked to a DNA polymorphism identified among related species through DArT. DArT is also able to outperform other genotyping techniques with polyploids due to the absence of primer competition found in other techniques. [2] Polyploids are commonly found among agriculturally important crops. [2] For example, DArT has been used to conduct genome-wide analysis among Musa species, which includes bananas and plantains, which led to the development of a phylogenetic cladogram based on genetic markers derived from DArT techniques. These developments enhance breeding knowledge to obtain desirable yields and products. [3]

Expedited recognition of markers found with genes responsible for phenotypes is also being studied in animals with the help of DArT. [2] [10] Mosquitoes’ resistance to insecticide has been linked to specific mutations in genes that confer resistance to certain species of mosquitoes over others. [10] Genotypic variations were found through markers while conducting DArT analysis on relevant samples.

Genomic mapping

Since DArT is able to find genetic relations among species within a metagenome in a cheap and expedited manner, it has been integral to developing physical and genetic maps of closely related species. [2] [8] In its inception, DArT was used to develop phylogenetic cladograms of rice subspecies based on the presence or absence of DNA fragments in each species’ genome. [1] In the same manner, DArT was incorporated in fabricating genetic maps for A. thaliana by conducting an automated version of DArT. [2] [9] Wheat, a hexaploid, is also another crop that has benefited from implementation of a DArT analysis as a Bacterial Artificial Chromosome (BAC) of the largest chromosome, 3B, was created from markers detected through DArT assays. [8] [11]

Related Research Articles

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In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

<span class="mw-page-title-main">Serial analysis of gene expression</span> Molecular biology technique

Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.

TILLING is a method in molecular biology that allows directed identification of mutations in a specific gene. TILLING was introduced in 2000, using the model plant Arabidopsis thaliana, and expanded on into other uses and methodologies by a small group of scientists including Luca Comai. TILLING has since been used as a reverse genetics method in other organisms such as zebrafish, maize, wheat, rice, soybean, tomato and lettuce.

<span class="mw-page-title-main">Amplified fragment length polymorphism</span>

AFLP-PCR or just AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990s by KeyGene, AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments. A subset of the restriction fragments is then selected to be amplified. This selection is achieved by using primers complementary to the adaptor sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments. The amplified fragments are separated and visualized on denaturing on agarose gel electrophoresis, either through autoradiography or fluorescence methodologies, or via automated capillary sequencing instruments.

<span class="mw-page-title-main">Representation oligonucleotide microarray analysis</span>

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<span class="mw-page-title-main">MAGIChip</span>

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Massive parallel signature sequencing (MPSS) is a procedure that is used to identify and quantify mRNA transcripts, resulting in data similar to serial analysis of gene expression (SAGE), although it employs a series of biochemical and sequencing steps that are substantially different.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

<span class="mw-page-title-main">Restriction site associated DNA markers</span> Type of genetic marker

Restriction site associated DNA (RAD) markers are a type of genetic marker which are useful for association mapping, QTL-mapping, population genetics, ecological genetics and evolutionary genetics. 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 site of a restriction enzyme throughout the genome. Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms mainly in form of single nucleotide polymorphisms (SNPs). Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers. Although genotyping by sequencing presents an approach similar to the RAD-seq method, they differ in some substantial ways.

<span class="mw-page-title-main">Combined bisulfite restriction analysis</span>

Combined Bisulfite Restriction Analysis is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. The technique is a variation of bisulfite sequencing, and combines bisulfite conversion based polymerase chain reaction with restriction digestion. Originally developed to reliably handle minute amounts of genomic DNA from microdissected paraffin-embedded tissue samples, the technique has since seen widespread usage in cancer research and epigenetics studies.

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<span class="mw-page-title-main">DNA nanoball sequencing</span>

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In the field of genetic sequencing, genotyping by sequencing, also called GBS, is a method to discover single nucleotide polymorphisms (SNP) in order to perform genotyping studies, such as genome-wide association studies (GWAS). GBS uses restriction enzymes to reduce genome complexity and genotype multiple DNA samples. After digestion, PCR is performed to increase fragments pool and then GBS libraries are sequenced using next generation sequencing technologies, usually resulting in about 100bp single-end reads. It is relatively inexpensive and has been used in plant breeding. Although GBS presents an approach similar to restriction-site-associated DNA sequencing (RAD-seq) method, they differ in some substantial ways.

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide a broad account of which cellular processes are active and which are dormant. A major challenge in molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how gene expression is regulated.

References

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