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Assignment: DNA polymorphism and its importance

Submitted to: Dr. Ashrad javeed Submitted by: Wajiha iram

Institute of Mycology and Plant Pathology

DNA Polymorphism and its Importance

INTRODUCTIONGenetic polymorphism is the existence of variants with respect to a gene locus (alleles), a chromosome structure (e.g., size of centromeric heterochomatin), a gene product (variants in enzymatic activity or binding affinity), or a phenotype. The term DNA polymorphism refers to a wide range of variations in nucleotide repeats, or single nucleotide variants and they provide the basis for direct physical analysis of genotype using molecular methods. Some of the DNA sequence polymorphisms, like our spore color example, occur within functional genes. DNA polymorphisms, however, have two advantages over conventional functional mutations. The first is that the sequence difference is detected directly and no functional phenotype need ever be associated with that sequence. DNA polymorphisms that have no known function are called anonymous loci. The second advantage of DNA polymorphisms is that they occur in a genome at a very high frequency. One reason for their high frequency is that although functional gene mutations are by definition limited to coding region. DNA polymorphism occurs to any DNA sequence, whether it contributes to a coding region or not. DNA polymorphisms that do not affect a phenotype are not subject to selection pressure, so the majority of DNA variation is located in non-coding regions. Up to 30% of fungal nuclear DNA is comprised of non-coding sequences (i.e., spacers, introns, and

various sorts of repeated sequence). Variations in any of these regions are detectable as DNA polymorphisms, but not detectable at all as functional mutations. As compare to fungi, in humans and other higher eukaryotes 95% of DNA can be comprised of noncoding sequence. It contributes to chromosome structure (e.g., the DNA telomeres and centromeres), otherwise has no known function. Because small fraction of DNA is involved in functional coding, most of these nucleotide changes do not have any effect on the phenotype of the organism. DNA polymorphisms are used in genetic mapping studies to identify DNA markers that are genetically linked to disease genes in the chromosomes in order to pinpoint their location. They are also used in DNA typing for identifying individuals, tracking the course of virus and bacterial epidemics, studying human population history and improving cultivated plants and domesticated animals, as well as for genetic monitoring of endangered species and for many other purposes.

Types of DNA polymorphisms:Essentially seven major types of DNA polymorphisms can be used as genetic markers: Single nucleotide polymorphism (SNPs): for example, substitutions, deletions, and insertions Restriction fragment length polymorphism (RFLPs): representing a subtype of single nucleotide changes that lead to changes at restriction sites. Amplified Fragment Length Polymorphisms (AFLPs) Short tandem repeat polymorphism (STRPs). Variable number of tandem repeat polymorphism (VNTR). Interspersed Repetitive DNA Polymorphism. Randomly amplified polymorphic DNAs (RAPDs)

Each of these types of DNA polymorphisms has an associated set of methods that has been developed for their analysis (Table 1).

1- Single nucleotide polymorphism (SNPs):Polymorphisms corresponding to differences at a single nucleotide position (e.g., substitution, deletions, and insertions) are called single base pair polymorphisms or more recently, single nucleotide polymorphisms.

Figure 1: Single nucleotide polymorphism showing DNA sequence variation that occurs when a single nucleotide (A, T, C or G) in the genetic sequence altered. In order for an altered sequence to be considered a SNP, it must occur in a least 1% of the population.

Most polymorphisms of this type have only two alleles (i.e., they are biallelic) and thus, they are sometimes referred to as biallelic markers. With only two alleles, the maximum heterozygosity for each SNP marker is only 50%, making them generally less informative than STRPs (which typically have multiple alleles and heterozygosities well over 70%). In general, maps consisting of SNPs need at least three times more markers than those containing STRPs at comparable resolution (Kruglyak 1997). SNP Distribution is not uniform for any of the three categories: Over a complete genome (1/3 in coding, 2/3 in non-coding). Over all the chromosomes (fewer SNPs in sex chromosomes). Over a single chromosome (SNPs often concentrated around a specific location).

Coding Region SNPs:Types of coding region SNPs Synonymous: the substitution causes no amino acid change to the protein it produces. This is also called a silent mutation Non-Synonymous: the substitution results in an alteration of the encoded amino acid. A missense mutation changes the protein by causing a change of codon. A nonsense mutation results in a misplaced termination. One half of all coding sequence SNPs result in non-synonymous codon changes. SNPs occur frequently in most genomes and have low mutation rate; features that make them desirable for use in building comprehensive genetic maps. For example, in the human genome, a SNP with heterozygosity greater than 30% occurs, on average, approximately every 1.3kb. The increased production of genomic sequence data in conjunction with improved methods for analysis are leading to the systematic generation of genetic maps consisting of SNPs.

Methods for the analysis of SNPs:A number of different methods are available for the analysis of SNPs (Table 1). Some can only be used to analyzed known SNPs, whereas others can be used both to identify previously unknown SNPs and to analyze known SNPs. Almost all techniques used to identifying unknown single nucleotide variations require gel based electrophoresis. Some exploit differences in the electrophoretic or chromatographic mobility between DNA variants. Examples include DGGE, SSCP analysis, heteroduplex analysis, TGGE and denaturing HPLC. Some are based on chemical or enzymatic cleavage of mismatched heteroduplex DNA fragments followed by gel electrophoresis to look for changes in fragment length. Examples include CCM, CDI, and T4 endonulease VII (enzymatic) cleavage of mismatch.

Other methods use various DNA sequencing based approaches. These include UNG mediated T scan sequencing and direct DNA sequencing. In general all techniques that can be used for the analysis of both known and

unknown SNPs are relatively labor intensive and difficult to automate. In addition, gel

Table 1: Method for analyzing DNA polymorphismPolymorphic type Analysis technique Basic methodology

SNP or insertion / deletion

Denaturing gradient gel Electrophoresis (DGGE) Single stranded conformational Polymorphism (SSCP) Heteroduplex analysis (HP) Temperature gradient gel Electrophoresis (TGGE) Cleavase fragment length Polymorphism (CFLP) Chemical cleavage of mismatch (CCM) Carbodiimide modification (CDI) Enzymatic cleavage of mismatch (ECM) UNG- mediated T scan Direct sequencing DNA chip resequencing Allele specific primer extention (GBA, TDI) Oligonucleotide ligation assay (OLA, DOL) Taqman- ASO

PCR / gel electrophoresis PCR / gel electrophoresis PCR / gel electrophoresis PCR / gel electrophoresis PCR / cleavase treatment/ gel electrophoresis PCR / chemical cleavage/ gel electrophoresis PCR / chemical modification/ gel electrophoresis PCR / enzymatic cleavage/ gel electrophoresis PCR / UNG treatment/ gel electrophoresis PCR / sequencing / gel electrophoresis PCR / hybridization/ fluorescence detection PCR / sequencing/ ELISA or fluorescence detection PCR /ligation/ ELISA or fluorescence detection PCR / nuclease cleavage/ ELISA or fluorescence detection Restriction enzyme digestion/ southern hybridization or PCR / restriction digestion/ gel electrophoresis PCR/ gel electrophoresis or primary extension/ mass spectrometery Restriction enzyme digestion/ southern hybridization PCR/ gel electrophoresis

Restriction fragment length Polymorphism (RFLP)

Tandem array length

STRP analysis Minisatellite (VNTR) analysis

Unknown probably SNPs

Randomly amplified polymorphic DNAs (RAPDs)

based methods are susceptible to problems caused by nonspecific products generated during PCR.

Methods developed specifically for the analysis of known SNPs generally do not involve gel electrophoresis. They offer tremendous advantage with respect to the throughput and potential automation. Such nongel based approaches combine PCR with an allelic discrimination reaction followed by product detection. In general, the allelic discrimination step is based on one of three approaches: allele specific hybridization allele specific primer extension with DNA polymerase allele specific ligation with DNA ligase. The reaction format largely dictated by the detection scheme used. Solid phase formats are utilized for colorimetric ELISA detection or fluorescence imaging, whereas homogeneous formats are used for FRET detection. Examples of detection schemes with solid phase format include DNA chip resequencing with fluorescence detection allele specific hybridization, genetic bit analysis with ELISA detection and OLA with ELISA detection. Examples of homogeneous detection scheme include the TaqMan-ASO assay (5 nuclease cleavage of hybridized allele specific probe), the TDI assay and DOL assay, all with energy transfer detection. Since all these methods have two level of specificity (PCR followed by an allelic discrimination reaction), they are generally not susceptible to problems caused by nonspecific PCR products. Additionally, the output from these assays is either positive or negative and can be readily scored in an automated fashion, thereby minimizing errors associated with human interpretation of results.

Pattern and distribution of SNP in fungi:Relatively little is known about the patterns and distributions of SNPs in nonmodel organisms, including most fungi. In human pathogenic yeast Candida albicans, there is an SNP frequency of ~1% per nucleotide (Jones et al., 2004). Similarly, a survey of seven genomic loci for 84 natural strains of the model yeast Saccharomyces cerevisiae from Asia has identified a total of 62 SNPs, yielding an SNP frequency of 2.05%per nucleotide (Ayoub et al., 2006). The SNP frequencies are higher in populations of several

opportunistic pathogenic yeasts, such as Candida parapsilosis (~3.4 %; Fundyga et al., 2004), and the species complexes of Candida guilliermondii (~6.3 %; Lan & Xu, 2006) and Cryptococcus neoformans (~20% per nucleotide; Xu et al., 2000). Analysing more strains should yield additional SNPs within the sequenced DNA fragments. Nevertheless, all the abovementioned fungi have SNP frequencies much higher than that in the human genome, in which SNPs are observed approximately once every 250 bp (Miller et al., 2005).

SNPs Applications:Single nucleotide polymorphisms are commonly used in: Pharmacogenomics Diagnostic genomics Functional proteomics Therapeutic genomics

2- Restriction fragment length polymorphism (RFLPs):The number and length of DNA fragments resulting from digestion of genomic DNA with a restriction enzyme vary according to the number of restriction sites along the DNA molecule and the distance between the restriction sites. Variation in a base sequence can create or destory a restriction site, giving rise to a detectable variation in the length of DNA fragment. Such genetic variation is known as restriction fragment length polymorphism (RFLP) and represents the first type DNA polymorphism that was identified with Southern blot method.

This technique has been applied widely to plant pathogens, but has particularly important in taxonomic studies of fungi. It has been used to differentiate isolates of

Phialophora gregata from different hosts, aggressive and nonaggressive isolates of Ophiostoma ulmi, and dictinct genetic populations with Clletotrichum gloeosporioides as well as to clearify taxonomic issues in fungal genera including Verticillium, fusarium, phytopthora, pythium, sclerotinia, and armillaria.( Rudra P. Singh, 1995).

Figure 2 Restriction Fragment Length Polymorphism (RFLP) resulting from b-globin gene mutation. In the normal cell, the sequence corresponding to 5th to 7th amino acids of the bglobin peptide is CCTGAGGAG, which can be recognized by the restriction enzyme MstII. In the sickle cell, one base is mutated from A to T, making the site unrecognizable by MstII. Thus, MstII will generate 0.2 kb and 1.2 kb fragments in the normal cell, but generate 1.4 kb fragment in the sickle cell. These different fragments can be detected by southern blotting.

Methods for the analysis of RFLP:Methods to enrich directly for hybridization probes that detect RFLPs have been developed that involve : use of genomic substraction techniques e.g., RDA or RFLP substraction . These approaches could make the development of RFLP based maps more attractive, especially for use with organisms where no genetic map exists.


approach for efficiently identifying RFLPs involves the use of

hybridization probes specific for moderately repetitive sequences. This strategy has been used successfully in mice with polymorphic insertion sites of retrovirus related sequences and in human being with minisatellite sequences (Table 1). In addition to analysis by stranded gel transfer hybridization procedures (i.e., Southern analysis), RFLPs can be analyzed by the restriction digestion of a PCR amplified DNA segment that contains the variably present restriction site. Of course, this requires some knowledge of the DNA sequence flanking that restriction site ( at least enough to design a PCR assay that amplifies a DNA fragment containing the site). Such sequence information is generally not available for most RFLPs. However, known sequence variants ( e.g, mutations) often disrupt or create a restriction site, allowing for the development of a convenient RFLP based assay that involves PCR amplification, restriction digestion and gel electrophresis (without subsequent Southern blot analysis).

Advantages of RFLPs:Advantages of RFLPs includes: the relatively low cost and simple methods associated with both their initial identification and subsequent analysis the abilty to use gene containing DNA segments (cDNA or genomic) as RFLP detecting probes generation (as a by-product) of single copy probes that are useful for numerous other purposes.

Disadvantages of RFLPs:Disadvantages of RFLPs includes: the difficulting in automating the methods used for either their initial identification or subsequent analysis

the inherent reliance on DNA probes that must be physically distributed in the case of a given RFLP ( in contrast to primer sequences that can be electronically distributed in the case of PCR based genetic markers)

need for large amounts of genomic DNA for detecting RFLPs by Southern blot analysis.

3- Amplified fragment length polymorphisms (AFLPs):Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction fragment lengths caused by SNPs or INDELs (Insertion/Deletion) that create or abolish restriction endonuclease recognition sites. AFLP is a powerful tool for comparative genome analysis, which is described by Zabeau & Vos in 1993. The procedure of this technique is divided into three steps: 1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments. 2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences. 3. Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern AFLP has originally been presented as a tool for strain typing purposes; it is also very well suited to simultaneously resolve isolates belonging to different species from each other. This is relevant in the case where the identification of a microorganism may be uncertain such as the species from the morphologically very similar. If not properly identified, use of such typing data could easily lead to false conclusions. In a way, AFLP can be considered the perfect PCR alternative to DNA-DNA re-association studies. Classical DNA-DNA re-association studies rely on sequence similarities. If two species

share a certain amount of sequence information, it is to be expected that they will also share a certain amount of similarity in banding patterns from an AFLP fingerprint. In fact, this has already been demonstrated for a variety of bacterial and fungal species.

FIGURE 3 An overview of the AFLP technology

Advantages: no sequence information is required the PCR technique is fast a high multiplex ratio is possible


Genetic Diversity Analysis Variety Identification Acceleration of Inbred Conversions Removal of Linkage Drag Linked Marker assay Development Seed and Plant Quality Assay Development Introgression Line Library Construction AFLPs can also be used to compare the hyper- and hypo-methylated regions of the genome using methylation sensitive and insensitive restriction enzyme isoschizomers facilitating the detection of possible epigenetic effects

Genetic Map Construction

4- Short tandem repeat polymorphism (STRPs):Short tandem repeat polymorphism (STRPs), also called microsatellite market, consist of a short sequence, typically from one or four nucleotides long that is tandemly repeated several times, and often characterized by many alleles. For example (CATG)n in a genomic region and is typically in the non-coding intron region. By identifying repeats of specific sequences, it is possible to create a genetic profile of an individual. There are currently over 10,000 published STR sequences in the human genome. STR analysis has become the prevalent analysis method for determining genetic profiles in forensic cases. STRP markers can be detected through the use of restriction endonucleases that cut on either side of the repeat, followed by Southern blotting and detection with a probe specific for the repeated sequence. Alternatively, PCR amplification with primers located just outside of the region containing the repeated sequence can be used to generate amplification fragments whose lengths reflect the number of repeats.

When tandem repeated sequences are replicated during cell division, the number of repeats can change. Variation in the number of tandem repeats will result in variable length DNA fragments that can be detected by Southern blotting or PCR, depending of the length of the tandem repeat. If the repetitive sequence involves more than 5 base pairs (generally around 10 to 15 base pairs), these polymorphisms are called variable number of tandem repeat (VNTR) and are visualized by Southern blotting. On the other hand, if the repetitive sequence is shorter, these polymorphisms are called short tandem repeats or microsatellites and are best visualized by PCR. The most frequents type of microsatellites are the dinucleotide repeats involving cytosine and adenosine (CA repeats), whose length vary approximately between 24 base pairs (12 repeats) and 80 base pairs (40 repeat). This type of polymorphism is illustrated in figure 4.

Size of DNA fragment (in bp)

Figure 4 Short tandem repeat polymorphism

The number of copies of tandem repeats is indicated by the number of boxes and determines the size of DNA fragment measured between the sites of the two primers. It is visualized by separating the PCR products by electrophoresis. These short tandem repeat polymorphisms are abundant, evenly distributed within the genome, and highly polymorphic, making them the most widely used markers to map genetic diseases and to study the molecular basis of multifunctional phenotypes

Microsatellites or STR"s are ubiquitously present in the genomes of many fungi including Aspergillus spp. STR are widely used for genetic mapping and strain typing purposes. Bart-Delabesse et al. (1998) reported the first application of microsatellites for A. fumigatus. These markers were obtained by screening genomic DNA libaries of A. fumigatus for suitable, microsatellite containing sequences, a process that proved quite laborious in the pre-genomics era. A panel of 4 dinucleotide repeats was selected that performed well in comparative genotyping experiments (Lasker 2002). Recently, based on genomic sequence data that has become available, de Valk et al. (2005) reported a novel set of 9 tandem repeats for typing A. fumigatus isolates, the so-called STRAf assay (STRs of A.fumigatus).

Advantages of STRs over traditional RFLP techniques: Discrete alleles from STR systems may be obtained due to their smaller size, which puts them in the size range where DNA fragments differing by a single base pair in size may be differentiated. Smaller quantities of DNA, including degraded DNA, may be typed using STRs. STR processing is rapid and abundant STR are available in the human genome. Discrete alleles allow digital record of data that in turn allows the automated analysis through a computer.

5- Variable number of tandem repeat polymorphism (VNTR):VNTRs are polymorphisms where a particular base sequence, often less than 20 bp, is repeated, from a few to more than 60, at a specific locus. These can be found on many chromosomes, and often show variations in length between individuals

Figure 5

Schematic of a Variable Number of Tandem Repeats in 4 alleles.

VNTR structure and allelic variationIn the schematic above, the rectangular blocks represent each of the repeated DNA sequences at a particular VNTR location. The repeats are tandem - they are clustered together and oriented in the same direction. Individual repeats can be removed from (or added to) the VNTR via recombination or replication errors, leading to alleles with different numbers of repeats. Flanking the repeats are segments of non-repetitive sequence (shown here as thin lines), allowing the VNTR blocks to be extracted with restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique and their size determined by gel electrophoresis. Variable Number of Tandem Repeats (VNTR) polymorphism has been identified in the ITS1 region of the rDNA in 13 strains belonging to a species of Nodulisporium producing the novel indole diterpene nodulisporic acid. The number of tandem repeats found in these isolates varies from 2, 4 or 5 repetitions, and produces three length morphs in the ITS1 rDNA, spanning a length difference of 217, 308 and 352 nucleotides.

Use of VNTRs in genetic analysis VNTRs were an important source of RFLP genetic markers used in linkage analysis (mapping) of genomes. Now that many genomes have been sequenced, VNTRs have become essential to forensic crime investigations, via DNA fingerprinting and the CODIS database. When removed from surrounding DNA by the PCR or RFLP methods, and their

size determined by gel electrophoresis or Southern blotting, they produce a pattern of bands unique to each individual. When tested with a group of independent VNTR markers, the likelihood of two unrelated individuals having the same allelic pattern is extremely improbable. VNTR analysis is also being used to study genetic diversity and breeding patterns in populations of wild or domesticated animals.

6- Interspersed Repetitive DNA Polymorphism:Interspersed repetitive DNA is a type of polymorphism where a particular base sequence is repeated throughout the genome (not in tandem, at a single locus). Only one copy of the sequence is located at any given locus, but that same sequence is observed at thousands of different loci. These sequences are sometimes referred to as satellite DNA Interspersed repetitive DNA is thought to make up somewhere between 5% to 20% of the human genome. The percentages and specific distribution patterns of these dispersed sequences is generally conserved within species, but varies between species. For this reason, interspersed repetitive DNA sequences have been useful in tracing evolutionary relationships .

7- Randomly amplified polymorphic DNAs (RAPDs):RAPDs can be used in a simple, inexpensive, and efficient fashion for identifying polymorphic markers and building genetic maps. RAPD- based approaches have been used for identifying polymorphic markers between inbred populations. Specifically, the use of RAPDs involves low stringency PCR of genomic DNA with a single, short Oligonucleotide primer. The products generated from different individuals are then subjected to gel electrophoresis and directly compared, with any reliable differences in the number and/ or size of products reflecting a polymorphism.

RAPD can be useful for detecting genetic differences within species (Williams et al., 1990, Parker et al., 1998, Sunnucks 2000). This technique has been used to investigate intra specific genetic variation in several fungi (e.g., Fegan et al., 1993, Moore et al., 2001). RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes.

RAPD markers have also been useful in other investigations of genetic variation among geographically distant populations of fungi. Li et al., (2001) compared RAPD markers in populations of western gall rust fungus (Endocronartium harknesii) collected across western and central Canada from two host pine species. Most of the genetic variation in E. harknesii occurred between the two host species, but within hosts there was more variation among geographically widespread locations than within locations.

Advantages of RAPD:The main advantages of RAPDs are The low cost (corresponding to a few short oligonucleotides and several PCR assays). Potential for rapid identification of numerous polymorphisms.

Disadvantages of RAPD:However the disadvantages are significant and include The lack of a molecular handle (i.e., the ability to directly access) on any of the resulting polymorphism The lack of predictability, In some cases, the lack of reproducibility of low stringency PCR The fact that the marker are scored dominantly.

This last feature reduces the amount of genetic information that can be readily obtained. Nevertheless, the use of RAPDs in some settings should be considered, since there is no more rapid and inexpensive methods for developing genetic markers or maps for an organism.

Importance of DNA polymorphism:1- Human population historyDNA polymorphisms provide important information in anthropology to reconstruct the evolutionary origin, global expansion, and diversification of the human population.

2- Improvement of domesticated plants and animalsPlant and animal breeders have turned to DNA polymorphisms as genetic markers in pedigree studies to identify, by genetic mapping; genes that are associated with favorable traits in order to incorporate these genes into currently used varieties of plants and breeds of animals.

3- History of domesticationPlants and animal breeders also study genetic polymorphism to identify the wild ancestors of cultivated plants and domesticated animals, as well as to infer the practices of artificial selection that led to genetic changes in these species during domestication.

4- DNA polymorphism as ecological indicators

DNA polymorphisms are being evaluated as biological indicators of genetic diversity in key indicator species present in biological communities exposed to chemical, biological, or physical stress. They are also used to monitor genetic diversity in endangered species and species bred in captivity.

5- Evolutionary geneticsDNA polymorphism are studied in an effort to describe the patterns in which different types of genetic variation occur throughout the genome, to infer the evolutionary mechanisms by which genetic variation is maintained, and to illuminate the process by which genetic polymorphism within species become transformed into genetic differences between species.

6- Population studiesPopulation ecologists employ DNA polymorphisms to assess the level of genetic variation in diverse population of organisms that differ in genetic organization (prokaryotes, eukaryotes, organelles), population size, breeding structure, or lifehistory characters, and they use genetic polymorphism within subpopulations of a species as indicators of population history, patterns of migration, and so forth.

7- Evolutionary relationship among speciesDifferences in DNA sequences between species is the basis of molecular phylogentics, in which the sequences are analyzed to determine the ancestral history (phylogeny) of the species and to trace the origin of morphological, behavioral, and other types of adaptations that have arisen in the course of evolution.

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