DNA Marker Techniques

Keywords

Genetic marker, DNA sequence, chromosome, variation, mutation, minisatellites, protein variation, genetic mapping.

Introduction

A genetic marker is a known location of a gene or DNA sequence on a chromosome which is used in the identification of individuals or species. It is described as a variation or mutation in a DNA sequence surrounding a single base-pair change or a long one, like minisatellites. Gene markers help in the identification of genetic characteristics like blood groups and protein variation.

Examples of genetic markers are single polymorphism nucleotides (SNPs), restriction fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), microsatellites, and copy number variants (CNVs), STRs, and indels.

Genetic markers play a key role in genetic mapping on all animal and plant genetics. With DNA-based markers, it is theoretically possible to exploit the entire diversity in DNA sequence that exists in any cross. Genetic markers are used to track the inheritance that can help link an inherited disease with the responsible gene.

A genetic marker is a landmark. The advent of next-generation sequencing (NGS) has revolutionized genomic and transcriptomic approaches to biology. These new sequencing tools are also valuable for the discovery, validation, and assessment of genetic markers in populations. The prospects for the use of DNA-based markers in marker-assisted selection are considered, along with likely future trends in gene mapping.

Techniques

Genetic markers are divided into two classes biochemical markers and molecular markers. Biochemical markers are used to detect the changes or variations at gene level like changes in proteins and amino acids.

Whereas molecular markers are used to detect the changes at DNA level like nucleotide changes such as duplication, deletion, or insertion etc. There are two modes of inheritance exhibited by these markers, they are dominant/recessive and co-dominant. Generally, co-dominant markers are more informative than the dominant markers. Some of the commonly used types of genetic markers are explained below.

RFLP (Restriction fragment length polymorphism)

RFLP is polymorphism observed individuals with variations in DNA sequence generated by restriction enzymes using specific endonucleases. Most RFLP markers are co-dominant. The principle is the creation or deletion of restriction enzyme caused by insertions or deletion of mutations serve as the main basis for RFLP.

RFLP markers are moderately polymorphic, highly locus-specific, highly reproducible, and are highly abundant in the genome and randomly distributed. RFLP analysis is practically a laborious, time-consuming, and technically demanding procedure not recommended for automation.

AFLP (Amplified fragment length polymorphism)

AFLP marker analysis is the combination of RFLP and PCR technologies that involves DNA digestion using restriction enzyme reaction and PCR amplification. This involves DNA fragments of size range 80–500 bps. The procedure involved in AFLP analysis is the isolation of high-quality DNA from tissues of the research organism.

AFLP are dominant markers and polymorphisms are detected as present or absent of electrophoretic DNA bands in polyacrylamide gels. The bands are recorded as present or absent based on sizes generated for each sample.

RAPD (Random amplification of polymorphic DNA)

RAPD polymorphism technique is the most convenient and widely used as it is easy and quick to assay. In this analysis, prior DNA sequence information is not an important requirement because random primers are commercially available. RAPD PCR fragment lengths usually range between 0.5 and 5 kb. RAPD is a dominant marker and data quality is limited.

RAPD analysis is locus non-specific, and the interpretation of electrophoretic gel patterns is quite confusing. As these bands consist of co-migration it is difficult to assign and analyze the problem. Recent modifications have improved the RAPD technique into more efficient marker methods like SCAR, SRAP and CAPS.

These improved marker variants of RAPDs overcome the associated disadvantages of RAPDs and complement the efficiency in the applications of the marker.

SSR Microsatellite polymorphism (Simple sequence repeat)

SSRs are simple sequences that arrange in tandem repeats of mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide motifs. The most frequently occurring motifs are the mono, di, tri, and tetra nucleotides.

SSRs constitute one of the classes of microsatellites. SSRs are locus-specific markers that are inherited in a co-dominant pattern, that helps in plant mapping and population genetic studies. SSRs are very informative but can be quite expensive in the marker discovery or development stage of DNA sequencing.

SNP (Single nucleotide polymorphism)

SNPs are genetic polymorphisms caused by positional changes in single base pair of DNA sequence in a species or different species. SNPs are the most frequently occurring form of DNA sequence polymorphisms in organisms. SNPs are commonly located in coding and non-coding as well as intergenic regions of genomes with a frequency of one SNP per every 100-300 bps of DNA. These markers are more attractive than other molecular markers for genotyping. SNP markers are characteristically biallelic and stable over many generations. SNP markers are amenable to automation, high throughput and, a popular and convenient technique for molecular plant breeding applications.

DArT (Diversity Arrays Technology):

DArT is a generic microarray-based hybridization, high-throughput, and reproducible approach for detecting the presence and absence of individual fragments for DNA fingerprinting analysis. A good quality genomic DNA of 50-100 ng amount is enough for purposes of DArT analysis.

The advantage of DArT is that it is very cost effective. DArT marker analysis is used to identify markers in the genomic library. DArT markers are scored with high accuracy and the DArT microarray platform itself allows flexibility of applications. DArT method presents many merits compared to other molecular marker technologies.

STS (Sequence tagged sites)

STS is a DNA section that is characteristically short and exhibits sequence uniqueness. The exact STS sequence is usually found only at one location in the genome. STS was found useful and adopted in plant genomic studies. STS markers are co-dominant, technically simple to carry out and highly reproducible. This technique has been applied to obtain successful results in species of several crops.

SSLP (or Simple sequence length polymorphism)

VNTR (or Variable number tandem repeat)

STR (or Short tandem repeat)

SFP (or Single feature polymorphism)

RAD markers (or Restriction site associated DNA markers)

Key Points

• These methods are used to help study the numerous populations of tens or hundreds of individuals for which genomic resources were not previously available using sequencing techniques are making new discoveries using thousands of genetic markers in sequencing in the whole genome.
• There is impact of several factors such as the availability of genomic resources, the levels of polymorphism, the pooling of samples and the choice of restriction enzyme on the design, and implementation of high-throughput marker discovery and genotyping experiments.
• As analysis of data from these methods is challenging sometimes, high-throughput marker data is explained for new methods of processing.
• This whole situation is likely to change in the next few years, as sequencing costs continue to fall rapidly. And these methods are more economical than whole-genome sequencing.

Conclusion

In this concept different types and techniques of DNA markers, their principles and methodology of the most popular and extensively used molecular markers have been explained. Some of the largest and most elaborate coverage of molecular markers have been seen above. The molecular marker techniques discussed comprise primarily the well-established Arbitrarily Amplified DNA (AAD) marker methods, the microsatellite-based marker techniques, and the retrotransposon-based molecular marker approaches. Indeed, molecular or DNA marker techniques provide tremendous opportunities for molecular genomic research. These markers should not be a substitute for biochemical markers rather, molecular markers should be applied as complementing techniques in genomics and plant breeding to enhance agricultural production, sustainable food, and nutrition supply.