Community Research and Development Information Service - CORDIS

FP5

HERGEN Report Summary

Project ID: Q5RS-2001-01370
Funded under: FP5-LIFE QUALITY
Country: United Kingdom

Micro-satellite calibration

Inter-calibration of microsatellite molecular genetic markers between partner institutes. Thirteen microsatellite loci were originally tested by the three partners involved in microsatellite DNA work (Partners 1a (DIFRES), 4 (UGOT), and 5 (UHULL)) on the same set of 40 standard individuals previously distributed among the 3 laboratories. Four of these were tetranucleotide loci developed for Atlantic herring by McPherson et al. (2001); eight were tetranucleotide loci developed for Pacific herring by Olsen et al. (2002), and one was a di-nucleotide locus isolated from Pacific Herring by O'Connell et al. (1998).

Different DNA extraction protocols (e.g., Phenol-Chloroform (Taggart et al. 1992), HotSHOT method (Truett et al. 2000), and Chelex resin (Walsh et al. 1991), as well as a broad array of different PCR amplification conditions were tested. DNA products were analysed using Pharmacia ALF-Express and MJ Research automated sequencing systems.

The following conclusions were reached:
- Chelex resin-based method was initially chosen as the extraction protocol, although this was later discarded in flavour of the HotSHOT method (Truett et al, 2000), since the former method yielded poorer quality of DNA. This allowed the PCR reaction to take place at more stringent conditions, so minimising the amplification of non-specific fragments.
- To ensure consistent genotyping and scoring across laboratories, single, unequivocal sizes were assigned to each allele in all loci.
- To ensure quality control of genotyping, two "standard heterozygote individuals" of known genotype were run alongside all samples, for each locus. The standards were chosen so as to cover a wide allelic range for each locus.
- To maximise consistency of genotyping, a procedure of "cross-check" scoring was adopted. The procedure involved the exchange between laboratories of 10 individuals to be scored blindly.
- Any "difficult-to-genotype" samples would not be scored in the first instance, but re-analysed and, if still "uncertain", ultimately recorded as missing values.

Use of these criteria permitted consistent scoring of alleles using 3 types of automatic sequencers situated in the 3 different laboratories, and would provide a valuable first step for other projects where microsatellite data from multiple sources is to be reliably combined.

McPherson et al. 2001. Molec. Ecol. Notes, 1: 31-32; O'Connell et al. 1998. Molec. Ecol. 7: 357-363; Olsen et al. 2002. Molec. Ecol. Notes, 2: 101-103; Taggart et al. 1992. J. Fish Biology, 40: 963-965; Truett et al, 2000 BioTechniques, 29: 52-54; Walsh et al. 1991. Biotechniques, 10: 506-513.

Contact

Bill HUTCHINSON, (Experimental Officer)
Tel.: +44-0148-2465536
Fax: +1-482-465458
E-mail
Record Number: 44175 / Last updated on: 2007-06-18
Information source: e-TIP
Collaboration sought: Available for consultancy, Further research or development support, Information exchange/Training
Stage of development: Scientific and/or technical knowledge (basic research)