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     minisatellites minisatellites Presentation Transcript

    • MINISATELLITES
    •  minisatellite (also referred as VNTR) is a sectionof DNA that consists of a short series of bases10–60 bp.[1][2] These occur at more than 1,000locations in the human genome. Minisatellites have been confused withMicrosatellites (also called as Short TandemRepeats or STRs). STRs are also repeatedsequences, but they are usually 2–13 nucleotideslong. The term minisatellite has beeninterchangeably used with variable numbertandem repeats (VNTRs).
    •  They are usually distinguished according to thesize of the repeats:Microsatellites, also termed Short tandem repeats(STRs) consist of tracts of repeats of 1-7bp, andminisatellites also called (VNTR) usually containrepeats of longer length (100-several hundred bp)Minisatellites can be found in tandem arrays, butthe majority are interspersed in the genome.Whilst microsatellites are found mostly in tandemrepeats.
    •  Minisatellites" consist of repetitive, generally GC-rich, variant repeatsthat range in length from 10 to over 100 bp. These variant repeats aretandemly intermingled, which makes minisatellites ideal for studyingDNA turnover mechanisms. Some minisatellites contain a central (or "core") sequence of letters“GGGCAGGANG” (where N can be any base) or more generally astrand bias with purines (adenosine (A) and guanine (G)) on one strandand pyrimidines (cytosine (C) and thymine (T)) on the other. It has beenproposed that this sequence encourages chromosomes to swap DNA.In alternative models, it is the presence of a neighbouring cis-actingmeiotic double-strand break hotspot which is the primary cause ofminisatellite repeat copy number variations. Somatic changes aresuggested to result from replication difficulties (which might includereplication slippage, among other phenomena). When such eventsoccur, mistakes are made, thus causing minisatellites at over 1,000locations in a persons genome to have slightly different numbers ofrepeats, thereby making each individual unique. The most highlymutable minisatellite locus described so far is CEB1 (D2S90) describedby Vergnaud
    •  Since the fortuitous discovery of the first humanminisatellite in 1980 by A.R. Wyman and R. White[4]and especially the discovery that the extremepolymorphism of minisatellites made them superb forDNA fingerprinting by Alec Jeffreys,[5] this class ofrepeats has been an intense focus of studies thathave addressed the turnover mechanisms thatprovoke their instability. Due to their high level ofpolymorphism, minisatellites have been extensivelyused for DNA fingerprinting as well as for geneticmarkers in linkage analysis and population studies. Minisatellites have also been implicated as regulatorsof gene expression (e.g., at levels oftranscription, alternative splicing, or imprint control) or
    •  Multi locus probes Repetitive DNA. A major step forward in genetic identification is thediscovery that about 30–90% of the genome of virtually all the species is constituted byregions of repetitive DNA, which are highly polymorphic in nature. These regions containgenetic loci comprising several hundred alleles, differing from each other with respect tolength, sequence or both and they are interspersed in tandem arrays ubiquitously. Therepetitive DNA regions play an important role in absorbing mutations in the genome. Of themutations that occur in the genome, only inherited mutations play a vital role in evolution orpolymorphism. Thus repetitive DNA and mutational forces functional in nature together formthe basis of a number of marker systems that are useful for various applications in plantgenome analysis. The markers belonging to this class are both hybridization-based andPCR-based. Microsatellites and minisatellites. The term microsatellites was coined by Litt andLutty26, while the term minisatellites was introduced by Jeffrey2. Both are multilocus probescreating complex banding patterns and are usually non-species specific occurringubiquitously. They essentially belong to the repetitive DNA family. Fingerprints generated bythese probes are also known as oligonucleotide fingerprints. The methodology has beenderived from RFLP and specific fragments are visualized by hybridization with a labelledmicro- or minisatellite probe. Minisatellites are tandem repeats with a monomer repeat length of about 11–60 bp, whilemicrosatellites or short tandem repeats/simple sequence repeats (STRs/SSRs) consist of 1 to 6 bp long monomer sequence that is repeated several times. Theseloci contain tandem repeats that vary in the number of repeat units between genotypes andare referred to as variable number of tandem repeats (VNTRs) (i.e. a single locus thatcontains variable number of tandem repeats between individuals27) or hypervariable regions(HVRs) (i.e. numerous loci containing tandem repeats within a genome generating highlevels of polymorphism between individuals2). Microsatellites and minisatellites thus form anideal marker system creating complex banding patterns by simultaneously detecting multipleDNA loci. Some of the prominent features of these markers are that they are dominantfingerprinting markers and codominant STMS (sequence tagged microsatellites) markers.Many alleles exist in a population, the level of heterozygosity is high and they follow
    •  Minisatellite and microsatellite sequences converted into PCR-based markers Sequence-tagged microsatellite site markers(STMS). This method includes DNA polymorphism using specific primers designed from the sequence data of a specific locus.Primers complementary to the flanking regions of the simple sequence repeat loci28 yield highly polymorphic amplificationproducts. Polymorphisms appear because of variation in the number of tandem repeats (VNTR loci) in a given repeat motif. Tri-and tetranucleotide microsatellites are more popular for STMS analysis because they present a clear banding pattern after PCRand gel electrophoresis29. However, dinucleotides are generally abundant in genomes and have been used as markers e.g.(CA)n(AG)n and (AT)n (ref. 30). The di- and tetranucleotide repeats are present mostly in the non-coding regions of thegenome, while 57% of trinucleotide repeats are shown to reside in or around the genes. A very good relationship between thenumber of alleles detected and the total number of simple repeats within the targeted microsatellite DNA has been observed. Thuslarger the repeat number in the microsatellite DNA, greater is the number of alleles detected in a large population31. Direct amplification of minisatellite DNA markers (DAMD-PCR). This technique, introduced by Heath et al.32, has been explored asa means of generating DNA probes useful for detecting polymorphism. DAMD-PCR clones can yield individual-specific DNAfingerprinting pattern and thus have the potential as markers for species differentiation and cultivar identification33. Inter simple sequence repeat markers (ISSR). In this technique, reported by Zietkiewicz et al.34, primers based on microsatellitesare utilized to amplify inter-SSR DNA sequences. Here, various microsatellites anchored at the 3¢ end are used for amplifyinggenomic DNA which increases their specificity. These are mostly dominant markers, though occasionally a few of them exhibitcodominance. An unlimited number of primers can be synthesized for various combinations of di-, tri-, tetra- and pentanucleotides[(4)3 = 64, (4)4 = 256] etc. with an anchor made up of a few bases and can be exploited for a broad range of applications in plantspecies. Other repetitive DNA-type markers Transposable elements. A large number of transposable repeat elements have beenstudied in plants; however, only a few have been exploited as molecular markers. In evolutionary terms, they have contributed togenetic differences between species and individuals by playing a role in retrotransposition events promoting unequal crossing over.Retrotransposon-mediated fingerprinting has been shown to be an efficient fingerprinting method for detection of geneticdifferences between different species. Alu-repeats. This strategy was developed to fingerprint genotypes using semispecific primers, complementary to repetitive DNAelements called ‘Alu-repeats’, in human genome analysis. Alu repeats are a class of randomly repeated interspersedDNA, preferentially used for Alu PCR as they reveal considerable levels of polymorphism35. These representatives of short andlong interspersed nuclear elements are known as SINES. Alu elements are approximately 300 bp in size and have been suggestedto be originated from special RNA species that have been reintegrated at a rate of approximately one integration event per 10000years. These repeats have been studied largely in humans, while their function in plants remains largely unexplored. Repeat complementary primers. As an alternative to the interspersed repeats, primers complementary to other repetitive sequenceelements were also successfully used for generation of polymorphisms, e.g. introns/exons splice junctions36, tRNAgenes37, 5sRNA genes38 and Zn-finger protein genes39. Primers complementary to specific exons, resulting in the amplification ofthe intervening introns have been studied by Lessa et al.40. One of the strengths of these new strategies is that they are moreamenable to automation than the conventional hybridization-based techniques