Microsatellite library creation
Finding existing microsatellites
The following database should be searched before any NEOF application is submitted requesting the development of a microsatellite marker set:
Genomic libraries are enriched for microsatellites as described by Armour et al. (1994) using modifications suggested by Gibbs et al. (1997) and Glenn & Schable (2005) and sequenced with an Illumina MiSeq Sequencer.
Unless specifically requested - and approved - libraries are created without the visitor present. This will take approximately 2-3 months. Visitors are then expected to attend the facility at Sheffield for training and to further develop the library.
Marker development can take (the visitor) 1-3 months and the genotyping of individuals using the validated markers can take another 1-3 months (full training is provided). This work involves the following techniques:
- DNA assessment
- digestion of the DNA using restriction enzymes
- library preparation
- enrichment of the DNA for repeat motifs
- visualisation of PCRed library products on agarose gel
- Illumina MiSeq sequencing
- sequence analysis
- primer design
- PCR amplification (visitor attends at this point)
- assessement of amplification and polymorphism using an ABI3730 (Applied Biosystems DNA Sequencer)
- allele assignment using GENEMAPPER software
- marker and data validation
- data analyses, such as analyses of population structure or parentage (as required, training provided).
Poor starting-quality DNA is the main cause of the failure of attempts to create enriched microsatellite libraries. For this reason, it is essential for users to provide DNA of a high quality. The following pointers have been listed.
- A DNA extraction protocol should be used that will produce DNA of the highest quality possible (eg Bruford et al. 1998).
- A minimum of 10 micrograms of high-quality DNA at a concentration of 0.1-0.25 micrograms per microlitre is required for creating an enriched microsatellite library.
- The quality of the undigested DNA should be checked on a 0.8% agarose gel. Good quality DNA is indicated by a high tight clear band, running at approx. 23kb. The band should be without fuzziness/ smearing around the band (so clear of DNA degradation) and not smeary along the length of the whole well lane (avoiding protein contamination). If RNA is present (usually seen as a wide band at 50bp), the DNA should be incubated at 37C for 1 hour with RNase (only if needed) and rechecked on a gel to confirm no RNA is visible (see Sambrook et al. 1989)
- The concentration should be checked on a spectrophometer and the 260:280nm absorbance ratio calculated to give a measure of the protein and RNA content.
- The DNA quality should be high and confirmed by checking it digests well with restriction enzymes such as MboI.
- High-quality DNA should also be confirmed by PCRing the DNA if any suitable (nuclear) primers exist, such as sex-typing primers.
- Copies of gel photographs and spectrometer/fluorimeter readings to indicate the quality and concentration of the genomic DNA should be emailed to the Facility Manager Deborah Dawson (D.A.Dawson@sheffield.ac.uk) to assess the DNA quality before it is shipped to Sheffield.
When the quality is confirmed, DNA should be posted or transported to the facility at Sheffield on dry ice (see our contact page for address details).
If posting next day delivery in the UK, then posting in a cool box is probably enough to protect the samples.
Armour JAL, Neumann R, Gobert S, Jeffreys AJ (1994) Isolation of human simple repeat loci by hybridization selection. Human Molecular Genetics, 3, 599-605.
Bruford MW, Hanotte O, Brookfield JFY, Burke T (1998) Multilocus and single-locus DNA fingerprinting. In: Molecular Genetic Analysis of Populations: A Practical Approach, 2nd edn, (ed. Hoelzel, A.R.), pp. 287-336. IRL Press, Oxford.
Gibbs M, Dawson DA, McCamley C, Wardle AF, Armour JAL, Burke T (1997) Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats. Animal Genetics, 28, 401-417.
Glenn TC, and N. A. Schable. 2005 Isolating microsatellite loci. Methods in Enzymology. 395, 202-222.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a Laboratory Manual, second edition. Cold Spring Harbor Laboratory Press, NY.
The NERC Environmental Omics Facility (NEOF)
Making state-of-the-art molecular genetics facilities and training available to the UK science community.