Question

Can you please answer these questions:

1. Define the Microsatellites? Why is there a high rate of the variability of microsatellites in cells?
2. What is Amplified Fragment Length Polymorphism?
3. What are the Characteristics of AFLP and its advantages?

Thank you in advance. It means a lot to me!!!

Answer 1

1. Microsatellites are simple sequence tandem repeats (SSTRs). The repeat units are generally di-, tri-, tetra- or pentanucleotides. For example, a common repeat motif in birds is ACn, where the two nucleotides A and C are repeated in bead-like fashion a variable number of times (n could range from 8 to 50). They tend to occur in non-coding regions of the DNA (this should be fairly obvious for long dinucleotide repeats) although a few human genetic disorders are caused by (trinucleotide) microsatellite regions in coding regions. On each side of the repeat unit are flanking regions that consist of "unordered" DNA. The flanking regions are critical because they allow us to develop locus-specific primers to amplify the microsatellites with PCR (polymerase chain reaction). That is, given a stretch of unordered DNA 30-50 base pairs (bp) long, the probability of finding that particular stretch more than once in the genome becomes vanishingly small (if the four nucleotides occur with equal probability then the probability of a given 50 bp stretch is 0.2550. In contrast, a given repeat unit (say AC19) may occur in thousands of places in the genome. We use this combination of widely occurring repeat units and locus-specific flanking regions as part of our strategy for finding and developing microsatellite primers. The primers for PCR will be sequences from these unique flanking regions. By having a forward and a reverse primer on each side of the microsatellite, we will be able to amplify a fairly short (100 to 500 bp, where bp means base pairs) locus-specific microsatellite region.
2. Microsatellite (or simple sequence repeat (SSR)markers are based on repeated sequences of one- to six-base core sequences (typically two to four), found interspersed in the genome. Since the sequences flanking the repeats are conserved, but the length of the repeat itself varies, these markers can be detected by PCR using a pair of primers flanking the microsatellite. Each microsatellite tags a single locus in the diploid genome. Microsatellites are particularly useful because of their: (i) abundance in genomes, (ii) high degree of variability in the repeat sequence, and (iii) reproducibility. The use of microsatellites for grapevine genetics includes: the identification of cultivars, the relatedness of cultivars and the analysis of the parentage of crossesAlthough microsatellite markers are a robust and useful tool in studying grapevine genetics, their development is time-consuming and expensive (prior to the draft grapevine genome sequence being released). The grapevine research community made a concerted effort since 2000, through the IGGP VMC, to develop microsatellite markers for the grapevine research community.

Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites.The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. AFLP principle After final amplification, selectively amplified fragments are separated by gel electrophoresis and visualized autoradiographically. MseI-MseI fragments are excluded from the autorad because only EcoRI-directed primers are normally labeled. Typically, the autorad has 100-300 fingerprints with sizes ranging from 80 to 500 nucleotides. Only a subset (10-40) of these total bands is polymorphic between two related individuals, such as Arabidopsis thaliana Columbia and Landsberg erecta ecotypes. Using 3-bp selective primer extensions gives 128 possible linker combinations. Therefore, 128 subsets of genomic DNA can be readily amplified. Thus, thousands of markers can be generated quite rapidly.

ADVANTAGES :-

AFLP is a technique used to detect polymorphisms in DNA when no information about the genome is known. Following restriction enzyme digestion of DNA, a subset of DNA fragments is selected for PCR amplification and visualization. A unique fingerprint is generated for a particular genome. AFLP®, first developed for plant studies, is now used for a wide variety of genetic analysis applications.

The power of AFLP analysis is the speed with which the technique can quickly generate large numbers of marker fragments for any organism, without the need for any sequence data.

CHARACTERISTICS:-

An AFLP band usually has two alleles in each locus (existence or absence of a certain band). The number of bands in a typical run of AFLP is several tens.