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Lecture 16, part 3 of 3
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So far, we've only been talking about one kind of molecular marker: RFLPs. In principle, any method that can identify mutations at any chromosomal location can be used to mark a site on a chromosome. RFLPs are time consuming and expensive.  PCR-based methods offer an alternative to RFLPs. PCR-based markers can be used for all the same purposes as RFLPs

F. Microsatellites (VNTR)

Microsatellites are regions of simple sequence tandem repeats. They lend themselves to length polymorphisms, most likely because of strand slippage during DNA replication. The net result is that it is easy to find polymorphic microsatellites in which different alleles have different numbers of repeat units, so that the total length of a given microsatellite locus may vary.



For a given locus containing a microsatellite, PCR primers specific for that locus are designed from the unique sequences flanking the repeats. Thus, microsatellite alleles are usually based on length polymorphism. Polymorphism at the priming sites would result in loss of bands, rather than changes in length.

Example: Aarnes SG et al. (2015) Identification and evaluation of 21 novel microsatellite markers from the automnal moth (Epirrita autumnata) (Lepidoptera: Geometridae). Int. J. Mol. Sciences 16:2241-22554. doi: 10.3390/ijms160922541.

The authors identify a number of microsatellite loci in the autumnal moth, which are polymorphic for the number of copies of short tandem repeats. Both alleles for each microsatellite locus were sequenced to determine the number of copies of tandem repeats in each, and the total length of the bands generated. For example, at locus A021 allele A contains 6 repeats of TGA, resulting in a PCR band whose total length is 92 bp, while allele B contain 7 copies of the TGA repeats, giving a PCR band of 95 bp.



Typically, several microsatellite loci can be amplified in a single PCR reaction. For each locus, primers for each locus are tagged with distinct flourescent dyes, so that the microsatellite bands for each locus flouresce at different wavelengths. PCR fragments are generated in separate reactions for each locus each using a different dye. After amplification, samples are mixed, and separated by capillary electrophoresis. This technique, rather than using a slab gel, runs DNA fragments through a thin capillary tube, containing polyacrylamide gel. A laser detector at the end of the capillaries detects the signals for each band at their characteristic wavelengths.


Results appear in a chromatogram, in which each DNA band appears as a peak. Bands for both alleles at each wavelength would fluoresce at the same wavelength. Homozygous loci, in which only one allele are present, give a single band eg. loci A019, A139 D005. Heterozygous loci, in which both alleles are present, give two bands eg. A021, A016, A022, A015.




Advantages of microsatellites
  • Highly-reproducible between labs
  • large number of polymorphisms per primer set
  • Often multiple alleles in a population, which can be highly-informative
  • Co-dominant
  • Disadvantages of microsatellites

    There are many different schemes for detecting polymorphism (ie.  differences in a given sequence among members of the population). Any of these can be used for molecular markers.

    G. How many markers must be screened to find one linked to a given trait?

    Regardless of the type of molecular marker employed, each assays a single genetic locus. That is, each marker assays a region of d cM on both sides of the marker. Another way to conceptualize it is to say that the genome is divided into G/2d segments of 2d each.




    What you might be tempted to do is to day that the numbers of markers to score, to cover the entire genome, is the genetic distance of all chromosomes added together (G),  divided by the genetic distance covered by each marker.

                       G
              N = -------------           
                     2d

    where

    N ::= the number of markers necessary to have at least one marker within d map units of any gene.

    As calculated here, N is referred to as "1 genome equivalent".

    The problem: N randomly chosen markers will be scattered unevenly across the chromosomes, so some regions will be full of markers, and other regions will not have any markers at all.


    Saturating the Genome with Markers

    The goal of any mapping project is to saturate the genome with markers.

    An initial map might be constructed using a relatively small number of markers. Since the chosen represent a random sampling of loci, they will be distributed unevenly across the genome. Consequently, some regions of each chromosome will be overrepresented in the map, and others will be underrepresented.  As we sample more markers and add them to the map, their map locations will also be unevenly distributed. Finally, if enough markers are used, there will always be at least one marker with in a certain distance d of any part of the genome.



    Therefore, it is necessary to screen a large number of markers before you find one that is linked to your gene.

    The following equation [Clark and Carbon (1976) Cell 9:91] allows us to calculate the number of markers necessary to find one that is linked to the gene of interest:
     
                     ln(1-P)
              N = -------------           
                     ln(1-f)

    where

    For example, to find a marker in the human genome (3615 cM) linked within 10 cM of any gene,
     
                                 ln(1 - 0.99)
              N = ------------------------------------- = 830 markers
                   ln [  1 -   (2 10 cM)/3615cM ]            (this is the 
        
                                                               bad news)

    The good news is

    H. Mapping kits.


    The examples above show that if you are trying to find a marker linked to a gene of interest, just screening randomly-chosen markers requires that a large number of markers be screened to be sure that at least one is linked to your gene.

    However, once a genome has been saturated with markers, you only need to search a small set of evenly spaced markers that together cover the entire genome. That way, no matter where your gene is, at least one of the markers you screen must be closely-linked with your gene.

    A mapping kit is a set of markers that are evenly spaced on the chromosomes. If you can define a minimal set of markers, you can detect linkage by testing a minimal number of probes.

    One way of looking at it is that now that we have saturated the genome, we can choose a set of evenly-spaced markers so that N = G/2d

    For example, if the human genome is 3615 cM in length, and we have markers evenly spaced at 20cM distance, then we need only  3615/20 = 181 markers to detect any gene.


    I. Summarizing molecular markers

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