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. Mutation process: Microsatellites are useful genetic markers because they tend to be highly polymorphic. It is not uncommon to have human microsatellites with 20 or more alleles and heterozygosities (Hexp = gene diversity, D) of > 0.85. Why are they so variable? The reason seems to be that their mutations occur in a fashion very different from that of "classical" point mutations (where a substitution of one nucleotide to another occurs, such as a G substituting for a C). The mutation process in microsatellites occurs through what is known as slippage replication. If we envision the repeat units (e.g., an AC dinucleotide repeat) as beads on a chain, we can imagine that during replication two strands could slip relative positions a bit, but still manage to get the zipper going down the beads. One strand or the other could then be lengthened or shortened by addition or excision of nucleotides. The result will be a novel "mutation" that comprises a repeat unit that is one bead longer or shorter than the original. The idea that adding or subtracting one repeat is likely easier than adding or subtracting two or more beads is the basis for using the Stepwise Mutation Model (SMM) as opposed to the Infinite Alleles Model (IAM). An advantage of the SMM (at least in theory) is that the difference in size then conveys additional information about the phylogeny of alleles. Under the IAM the only two states are "same" and "different". Under the SMM we have a potential continuum of different similarities (same size, similar in size, very different in size). If, however, the SMM does not hold, then we may be worse off using it -- it may actually be highly misleading. Even if the underlying mutation process is largely stepwise, it is not difficult to see how drift might affect the distribution of allele sizes in a way that would almost entirely invalidate the SMM (visualize this by examining Figs. 6.1 and 6.2 in Lecture 6). Show Advantages of microsatellites as genetic markers:
Codominant (heterozygotes can be distinguished from homozygotes, in contrast to RAPDs and AFLPs which are "binary, 0/1") PCR-based (means we need only tiny amounts of tissue; works on highly degraded or "ancient" DNA) Highly polymorphic ("hypervariable") -- provides considerable pattern Useful at a range of scales from individual ID to fine-scale phylogenies We are interested in conducting a genetic analysis of Species X using microsatellites, because we decide that microsatellites will provide the most information per unit effort and cost. How do we go about developing primers? If someone has developed primers for a closely related species, those primers will be well worth checking in our species. If, however, no primers have been developed for related species, we may need to develop our own. We do so by a sequence of steps: 1) Extract DNA from tissue (wide variety of possible methods depending upon tissue type) 2) Fragment the genome. Cut our genomic DNA into suitable size fragments with restriction enzymes. Generally, restriction enzymes that produce mean fragment sizes in the range of 300-600 bp are the desired goal. 3) Insert. Insert the fragments into plasmids. This step allows cloning of the fragments -- producing many copies of the 300-600 bp pieces we have inserted in the plasmids. To get a slightly more detailed idea of how plasmids act as cloning vectors, look up the boldface terms in the glossary of terms page. PUC19 is a commonly used plasmid for this sort of analysis. Why PUC19? The restriction sites in PUC19 are known (so that the ligated DNA fragments can later be cut out) and it replicates well in a bacterial culture. 4) Plate the plasmids on a nylon membrane. 5) Probe the membrane with labeled oligonucleotides of desirable repeats (e.g., AC10). 6) Culture the positive clones (the plasmid-fragments that bonded with the oligo probes). 7) Cut the insert out of the plasmids with restriction enzymes and run them out on an agarose gel. 8) Probe. Use Southern transfers to probe the digest again with labeled oligos. This serves:
b) to allow us to estimate the size of the insert. 10) Select. Analyze the sequence to check for "good" primer sites and useful repeat length (generally at least 8 repeats and it is often best to have more -- depending upon our intended application we may want long pure repeats or we may be interested in shorter interrupted repeats, which may have lower mutation rates). Criteria that enter into primer selection include:
c) avoidance of primer initiation sites that won�t bind well, avoidance of palindromes (sequences that have the same sequence from either end) and a number of others. d) total amplified product lengths of 100-250 bp, so that they are feasible for the sequencing gels or automated genotypers we will use for visualization. e) avoidance of repeats near end of sequenced region. Some of the positive clones we have sequenced may have good repeat units, but be too close to the end of the sequence. We then lack enough flanking region with which to design a primer. That, in part, is why we want fragments of 300-600 bp -- short enough to be feasible for sequencing, but long enough to reduce the likelihood that the repeat will be a "cliff-hanger." 11) Order the locus-specific primers (generally these will be 20-30 bp sections of the flanking regions not immediately adjacent to the repeat unit). Here is an example of a microsatellite sequence for scrub-jays that contains a repeat unit and forward and reverse primer sites. From beginning of forward primer to end of reverse primer, the above is 131 bp Repeat is CA11 Strassmann et al. (1996) has a more detailed run-through of much of this section. Screening existing microsatellite primers has been a major focus of research in my lab. Past projects include those of Sam Wisely (now on the faculty at Kansas State University; genetics of black-footed ferrets and other mustelids), Nicole Korfanta (genetic structure of migratory vs. resident populations of burrowing owls) and Marni Koopman (genetic structure of Boreal Owls). We may do a quick a guided tour of the laboratory procedures from DNA extraction from tissue (hair, blood, muscle etc.) to visualizing the amplified DNA on an ABI automated DNA sequencer in the Nucleic Acid Exploration Facility (NAEF). Here are the basic steps: 1) Extract the DNA. One often begins by somehow breaking up the tissue (e.g., by grinding in liquid nitrogen). Alternatives for the extraction process include classic phenol-chloroform extractions, salt-based extractions, and a variety of commercial kits. We are getting rid of proteins and other non-DNA tissue components in this step. A typical analysis might include extracting DNA from each of the individuals in a local population of 30 individuals. 2) Amplify. We add a very small amount of each of our 30 samples of extracted DNA to a PCR cocktail for amplification in a thermocycler. This is a "magic" step that has revolutionized molecular biology. We start with almost no DNA and wind up with enough that we can see it on a gel! Various "cocktail" recipes exist -- they typically contain the thermophilic bacterial enzyme Taq polymerase (essential), the dNTP mix (nucleotides that will allow massive replication of our target DNA), magnesium chloride, and the fluorescently labeled dNTPs (these will bind to the specially added M13 or T3 tail and light up under the laser and make bands of DNA alleles show up on the gel). 3) Load. We load our 30 amplified products in separate lanes in a large vertical polyacrylamide gel. We also load several lanes with a DNA ladder -- known-size fragments of amplified DNA of known quantity/concentration. A common ladder is lambda phage cut with restriction enzymes to yield a series of fragments. The newer capillary sequencers don't use a gel. 4) Run the sequencer. We run the amplified product through the sequencer until all the alleles have had time to run by the laser, which illuminates the fluorescent nucleotides and makes bands light up on the gel (or go digital-direct to the computer). The sequencer generates both an analog image (for older, gel-based sequencers) and digitally stored data concerning the size of the fragments. 5) Optimize (variations on Steps 2-4). It
often takes considerable fiddling to get the PCR conditions right for a particular combination of primer, DNA, thermocycler and sequencer. Major variables in optimization include: Alternative methods of visualization include "hand-built" polyacrylamide sequencing gels with silver-staining, CyberGreen staining, ethidium bromide staining or radioactive labeling. Many of these involve nasty chemicals (EtBr) or radioactivity, so we feel fortunate to be using a relatively clean, safe procedure. Fig. 8.1.
Stylized diagram of an electrophoretic gel for microsatellites. A current draws amplified DNA down Fig. 8.2. Representative microsatellite and gender
probe gel. DNA was amplified by PCR and run out on a Li-Cor E. How do we analyze the allelic information? For a slightly more detailed description go to the Genetic analysis page.
When two or more units are repeated in a sequential repetition?Chap 8. Which design principle refers to units?Progression. Which design principle refers to units that are opposite , creates variety and stimulates interest. Contrast. Balance in a design is identified by. A state of equilibrium between contrasting , opposite or interacting elements in the design.
Which design principle refers to units that are opposite creates variety and stimulates interest?Which design principle refers to units that are opposite, creates variety and stimulates interest? Contrast. Balance in a design is: A state of equilibrium between contrasting, opposite or interacting elements in the design.
What is elongated and angular face shape?People with rectangular faces have elongated angular lines with equally wide cheekbones, forehead, and jawline.
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