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Microsatellite Genotyping

 

What are microsatellites?

Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable in populations of DNA and within the alleles of an individual. The sequence below has a 20 dinucleotide repeat (40bp) stretch of CA that is shown in bold.

CGTTCAATAAGCAAAAATCCATAGTTTTAGGAATGTGGGCT
GCTTGGTGTGATGTAGAAGGCGCCAATGCATCTCGACGTAT
GCGTATACGGGTTACCCCCTTTGCAATCAGTGCACACACAC
ACACACACACACACACACACACACACACACAGTGCCAAGCA
AAAATAACGCCAAGCAGAACGAAGACGTTCTCGAGAACACC
AGAAGTTCGTGCTGTCGGGGCATGCGGCGAGTAAAGGGGAT

If you flank a microsatellite with fluorescent PCR primers then amplification will give a pair of fluorescent allelic products which will vary in size according to their repeat length. A population might possess 5 alleles which vary in size like this.

 

Figure 1

What is genetic linkage?

Genetic linkage is the tendency of two allelic features to be inherited together as an intact unit. Linkage is based upon an analysis of families. When features are close together on a chromosome there is a higher likelihood that they will be inherited together and familial recombination events will not have interfered. The likelihood that features will be inherited together is measured as a log of odds ratio (LOD) e.g. 100:1 or a LOD of 2. The statistical robustness of measuring linkage depends on testing many families so that one can really determine whether features are inherited together because they are linked or whether they are inherited together by chance alone.

 

Err... you said "recombination event", whats that?

A genetic recombination event is a crossover between the alleles of one of your parents. Chromosomes occur in pairs and in each pair; one will have come from your Mother and one from your Dad. If we consider a pair of chromosomes from our parents as being represented by the following, where capital letters represent the chromosome that Dad got from his Dad, and lower case from his Mum, and numbers represent the chromosome Mum got from her Dad and roman numerals the chromosomes she got from her Mum.

Child 1 gets two un-recombined chromosomes, but child 2 inherits a recombination event on both chromosomes.

 

Dad   A a B b C c D d E e F f G g H h I I J j

Mum   1 i 2 ii 3 iii 4 iv 5 v 6 vi 7 vii 8 viii 9 ix 10 x

Child 1   A i B ii C iii D iv E v F vi G vii H viii I ix J x

Child 2   a 1 b 2 c 3 D 4 E 5 F 6 G vii H viii I ix J x

So what's linkage disequilibrium?

When two features occur together as an intact unit within a population we say the features are in linkage disequilibrium. Features that occur predictably and randomly are in linkage equilibrium. Typically features in linkage disequilibrium are much closer together than those that are linked within families because in every generation since the features first occurred together there will have been an opportunity for them to become unlinked by genetic recombination thus spoiling the subsequent linkage disequilibrium.

 

So how can microsatellites be used in genetic studies?

Microsatellites can be used as markers in genetic studies of linkage in families and linkage disequilibrium studies of populations. In linkage studies one can examine large number of families and see when the alleles of specific markers are inherited together with a phenotype in more cases than not. Microsatellite repeat are amplified with fluorescently labeled primers and then the alleles from each individual in a family are separated by size and the marker tested for linkage with another. The raw family data looks like this:

 

Figure 6: Raw genotyping data.

Markers in a genome screen must be no more distant from the underlying genetic cause of the phenotype than the detectable level of marker - marker linkage. This is generally about 10Mbp and so 300 or more microsatellites are required to screen the entire genome for a disease gene using linkage. Linkage is a crude tool and refining the position of a disease gene is impossible since linkage will be quickly maximized.

In studies of linkage disequilibrium (LD), a marker allele is associated with a phenotype across an entire population. The spacing of the markers has to represent the level of LD across the genome (or locus) of the population. However, LD is not a regular phenomenon and it can extend from a few KB to many hundreds of KB. Therefore deciding where to place markers is problematic and a genome-wide screen of LD might require 500,000 markers or more to be typed per DNA. Therefore, genome-wide LD scans are not often performed and LD is more often reserved for analyzing just a few loci following a positive genome-wide linkage scan.

 

Why use microsatellites rathers than SNPs?

SNPs are great genetic markers but because of their low heterozygosity (the likelihood that a marker in any individual will appear heterozygous) you need to type lots of them. Microsatellites are good markers for studies of genetic linkage because they have a high heterozygosity. They are highly mutable markers with often 15 or more alleles in any given population. This means that allelic identity-by-descent can be readily established (unlike with bi-allelic SNPs) and linkage determined. However, the mutability of microsatellites can also prove problematic. Occasionally alleles can be seen to mutate within a generation giving rise to apparently non - Mendelian inheritance. This mutability becomes more of a problem when considering allelic associations within populations and it has been argued that SNPs offer a better chance of identifying marker-marker or marker-phenotype LD.

 

What about study design and power?

The maximum power to detect a genetic locus using linkage is restricted by the family material that one chooses to type. Linkage is refined by familial recombination and this occurs randomly. Statistical simulations of linkage studies can be made in order to determine the maximum outcome of these things and in turn whether to proceed experimentally with projects. Source BioScience LifeSciences can help you with these analyses before you commit to genotyping. Contact us us for further information.

Tell me about genome scans using microsatellites and linkage?

A genome scan is a series of genome-wide microsatellite genotypes across a set of families. In the first instance enough markers should be typed to cover marker-maker linkage in a step-wise manner across the entire genome. Historically, we have done this by typing an established set of 400 markers that we purchase from Applied Biosystems (LMS2.5 10cM) although a denser set of markers is also available (LMS2.5 5cM). For the majority of genome screens these markers will show marker-maker linkage, however there may be loci where random recombination has restricted linkage and further markers are required. Following this genetic linkage analysis of the phenotype-genotype can begin and LOD scores established.

 

How much DNA do you need per genome scan or per reaction?

DNA should be supplied at a standard concentration and in a microtitre plate. We need about 5ug of each DNA for a genome scan although we might require more or less depending on how simple we find the amplification. Customers always have the option of having unused material returned to them when their experiments are completed.

 

How long does this all take?

Microsatellite genotyping projects can take months or just a few days. The big projects get scheduled into a vacant spot in our calendar and small projects are often combined together so that we continue to offer the best value for money to all our customers. In both cases there can be a wait of a few weeks before your experiments begin. The sooner we get things scheduled the better.

 

What about after the genome scan?

Data is delivered in an agreed format by CD-ROM. The post-laboratory work is in two phases, some customers want us to do both, others prefer to do all this work themselves. The first phase of work is making the genotyping calls from the raw data; the second phase is the statistical genetic analysis of the results. The agreed price will reflect the level of analysis required.

 

Will you make custom microsatellite genotyping?

Some customers come to us after making a genome screen and ask for a set of fine mapping microsatellite markers. However, if the established information content is already near maximum then it might be detrimental to type further markers and perhaps impossible to obtain a legitimately higher LOD score. Fine mapping markers can be used to determine allelic associations or to confirm other genetic observations. However, one should always remember that the more markers typed, the more likely an apparent statistical significance will be found purely by chance alone.

 

What instrumentation have you got?

We use Matrix PlateMate plus for high throughput liquid handling and a Kbiosystems Super Duncan thermal cycler. We use an ABI Prism 3100 genetic analyzer for product analysis but for very high-throughput analysis we have the option of using ABI Prism 3730 genetic analysers. All work is tracked using our in-house LIMS system.

 

How much does a genotype cost?

A good figure to use in your estimations is £1 to £2 per genotype including genotype calls but not including genetic analysis. This figure is not a universal quotation; rather it is just a good place to start. Genotyping sometimes works out more expensive than this but more often it is cheaper for a genome scan. Please contact us with the precise details of your project and we will provide a written custom proposal.

 

How do I go about doing this research at Source BioScience LifeSciences?

This is not a something into which you should enter lightly. Source BioScience LifeSciences will not accept liability for the costs of genotyping projects that are subsequently shown to be scientifically weak even if you have consulted us during the design of your project. We assume that most genome scans are funded from a peer reviewed grant application and as such will have had expert consideration. After receiving your funding and when considering outsourcing this work to Source BioScience LifeSciences we encourage potential collaborators to visit us and discuss the requirements of their project directly so that our proposal can be tailored to your exact requirement. Contact us to arrange an appointment. When your project begins you will have direct access to a project manager that will manage your project at Source BioScience LifeSciences.

 

I've still got questions, who can I speak to?

OK, contact us now.