**5. The major technique for DNA-based molecular marker detection**

Different forms of DNA-based molecular markers can be tracked using a variety of techniques. Some of these techniques include RFLPs with Southern blots and polymerase chain reactions (PCRs). Recently great advances in methodology for DNA polymorphisms detection using real time PCR, hybridization techniques using DNA microarray chips, genome sequencing each technique has its own advantage and disadvantage.

#### **5.1. Restriction fragment length polymorphism with southern blot**

DNA digestion with restriction enzyme endonuclease cuts DNA at a specific sequence pattern known as a restriction endonuclease recognition site. Thus, the alleles differ in length and can be distinguished by gel electrophoresis, which can arise from a number of genetic events including point mutation in restriction sites, mutation that creates a new restriction site, insertion, deletion, and repeated sequences. The first polymorphic RFLP was described in 1980. RFLPs were the original DNA targets used for human identification, parentage testing, and gene mapping.

The method of hybridization of DNA with probes is called Southern blotting, after the name of the inventor, Southern [41]. RFLP requires relatively large amounts of DNA. Hence, it cannot be performed with the samples degraded by environmental factors and also takes longer time to get the results [42, 43]. PCR-RFLP is now replaced to avoid using Southern blot.

#### **5.2. Polymerase chain reaction**

Depending on the search algorithm, there are approximately 700,000–1,000,000 microsatellite loci which are 2–6 bp long in the human reference genome [33, 34]. Di- and tetra-nucleotides constitute about 75% of microsatellites, with the remaining loci containing tri-, penta, and hexanucleotide. Within genes, STRs are nonrandomly distributed across protein-coding sequences, untranslated regions (UTRs), and introns. STRs containing dinucleotide repeat units that are much more abundant in the regulatory or UTR regions than in other genomic regions. In the coding regions of the genes, repeats mostly have either trimeric or hexameric repeat unit, likely as a result of selection against frameshift mutations [34, 35]. "The mutation

per cell generation which is 10- to 10<sup>5</sup>


and 106

than the average mutation rates observed in nonrepeated regions of the genome"[36, 37].

"Polymorphism of tandem repeats within protein-coding regions reveals that tandem repeat variation is an important source of variation in many proteins, many of this variation is of significant impact on protein function. Tandem repeats has been associated with a number of diseases and phenotypic conditions, changes in the protein products of genes, leading to diseases, other tandem repeat polymorphisms in noncoding regions are known to modify function through their impact on gene regulation". "These polymorphisms can arise from events such as unequal crossover, replication slippage or double-strand break repair" [38].

Variations in the STR length play a significant role in modulating gene expression and STRs are likely to be general regulatory elements; regulatory STRs manifest significant polymor-

There are examples for distinctive phenotypic changes and diseases that are directly associated with the increases or decreases of microsatellite repeat arrays; for example, considering Huntington disease gene, triplet nucleotide mutations, the mutation that causes the disease, is an expansion of CAG repeats from the normal range of 11–14 copies to abnormal range of at least 38 copies. The extra CAG repeats that causes extra glutamine is produced [9] and there are more than 40 neurological diseases in humans, such as spinocerebellar ataxia with polyglutamine tracts, which are caused by microsatellite motif length changes in trinucleotide arrays [39]. Testing candidate genes for polymorphisms in exons, promoters, splice sites, or other regulatory regions will have to be done using SNP testing, because it is the most common polymorphisms and more likely responsible for phenotypic variations. For complex phenotypic traits and candidate loci, single-loci SNP analyses present less information due to the bi-allelic nature of the markers, as compared to the multi-allelic microsatellites. However, performing haplotype frequency may improve the accuracy [40]. Recently, polymorphic tandem repeated sequences and coy number variations have emerged as important sources of genomic diver-

rates of STRs often lie between 103

30 Genetic Diversity and Disease Susceptibility

phism because of their high intrinsic mutation rate [15].

sity that facilitate the study of genetic variations in health and diseases.

**5. The major technique for DNA-based molecular marker detection**

Different forms of DNA-based molecular markers can be tracked using a variety of techniques. Some of these techniques include RFLPs with Southern blots and polymerase chain reactions (PCRs). Recently great advances in methodology for DNA polymorphisms detection using In-vitro amplification of particular DNA sequences with the help of specifically chosen primers and DNA polymerase enzyme is done. The amplified fragments are separated electrophonically and detected by different staining methods. Real-time PCR useful modification of PCR can detect polymorphisms by various methodologies using real-time PCR chemistries, for example, TaqMan assay or molecular beacons.

#### **5.3. Genomic array technology**

Genomic array technology is a type of hybridization analysis allowing simultaneous study of large numbers of targets or samples. In 1987, macroarray evolved into the microarray. Tens of thousands of targets can be screened simultaneously in a very small area. Automated depositing systems (arrayers) can place thousands of spots on glass substrate of the size of a microscope slide (chip) with spotting representative sequences of each gene in triplicate, simultaneous screening of the entire human genome on a single chip. This technique facilitates the process of identifying specific homozygous and heterozygous alleles, by comparing the disparity of hybridization of the target DNA with each redundant probe. Microarray is also used to characterize genetic diversity and drug responses, to identify new drug targets, and to assess the toxicological properties of chemicals and pharmaceuticals [44].

#### **5.4. Sequencing**

Since technologies for rapid DNA sequencing have become available they are now widely used. There is a great progression for the detection of single nucleotide variants (SNVs) by direct sequencing, but intermediate-sized (from 50 bp to 50 kb) structural variants (SVs) remain a challenge. Such variants are too small to detect with cytogenetic methods but too large to reliably discover with short-read DNA sequencing. Recent high-quality genome assemblies using long-read sequencing have revealed that each human genome has approximately 20,000 structural variants, spanning 10 million base pairs, more than twice the number of bases affected by SNVs. New long-read sequencing approaches are needed to meet this challenge, as short-read sequencing technologies only detect 20% of the SVs present in the human genome [45–48].

Polymorphic STRs, together with SNPs and CNVs, can explain variability in response to pharmacotherapy because of their prevalence in the human genome and their functional role as regulators of gene expression and its applications. Pharmacogenetics is the study of the influence of genetics factors on drug response and metabolism. The science of pharmacogenetics when applied can be used to evade adverse drug reactions, predict toxicity and therapeutic

DNA Polymorphisms: DNA-Based Molecular Markers and Their Application in Medicine

http://dx.doi.org/ 10.5772/intechopen.79517

33

Establishing an individual's identity is one of the uses of DNA sequence information that highlights uniqueness of a particular sample [5], also known as genetic fingerprinting; DNA typing and DNA profiling are molecular genetic methods that enable the identification of individuals using hair, blood, semen, or other biological samples, based on unique patterns in their DNA. This uniqueness in each individual is the basis of human identification at the DNA level, forensic identification, determination of genetic variation, determination of family relationship, and one important instance is identifying good genetic matches for organ or marrow donation. When first described in 1984 by British scientist Alec Jeffreys, the technique used was minisatellites; these sequences are unique to each individual, with the exception of identical twins. Different DNA fingerprinting methods exist, using either restriction fragment length polymorphism (RFLP) or PCR or both. More than 200 RFLP loci have been described in human DNA. Initially, forensic medicine used minisatellite testing; however, this method requires a large amount of material and yield low-quality results especially when only little amount of materials are available. Nowadays, in most forensic samples, the study of DNA is usually performed by microsatellite analysis. The most useful microsatellite for human identification is those with a greater number of alleles, smaller size, higher frequency of heterozygotes (higher than 90%), and low frequency of mutations [43]. Among others, the microsatellite DNA marker has been the most widely used, due to its easy use by simple PCR, followed by a denaturing gel electrophoresis [40]. Each person has some STRs that were inherited from the father and some from mother, useful in paternity testing but however no person has STRs that are identical to those of either parent. The uniqueness of an individual's STR provides the scientific marker of identity and hence is helpful in forensic identification [54]. Genomic and mitochondrial are two types of DNA which are used in forensic sciences. The genomic DNA is found in the nucleus of each cell in the human body and represents a DNA source for most forensic applications. Mitochondrial DNA (mt DNA) is another source of material that can be used; various biological samples such as hair, bones, and teeth that lack nucleate cellular materials can be

"Majority of the length of the human Y chromosome is inherited as a single block in linkage from father to male offspring as a haploid entity. DNA genetic markers on the human Y chromosome are valuable tools for understanding human evolution, migration and for tracing relationships among males" [43, 56]. "Chromosome X specific STRs is used in the

failure, and refine therapeutic efficiency and improve clinical outcomes [53].

**7. DNA fingerprinting and human identification**

analyzed with mt DNA [43, 55].

**7.1. Sex-chromosome STR testing**
