Please use this identifier to cite or link to this item:
https://doi.org/10.1186/1471-2164-10-125
Title: | A simple and efficient method for isolating polymorphic microsatellites from cDNA | Authors: | Yue, G.H Zhu, Z.Y Wang, C.M Xia, J.H. |
Keywords: | complementary DNA deoxyribonuclease oligonucleotide complementary DNA microsatellite DNA animal tissue article Asian seabass biotinylation cloning vector controlled study DNA isolation DNA library DNA polymorphism DNA sequence fish gene frequency gene number heterozygosity intermethod comparison magnetic field microsatellite marker molecular cloning nonhuman nucleotide sequence allele animal bass expressed sequence tag gene library genetic marker genetics methodology molecular genetics sequence alignment Pisces Vertebrata Alleles Animals Bass DNA, Complementary Expressed Sequence Tags Gene Library Genetic Markers Microsatellite Repeats Molecular Sequence Data Sequence Alignment Sequence Analysis, DNA |
Issue Date: | 2009 | Publisher: | BioMed Central Ltd. | Citation: | Yue, G.H, Zhu, Z.Y, Wang, C.M, Xia, J.H. (2009). A simple and efficient method for isolating polymorphic microsatellites from cDNA. BMC Genomics 10 : 125. ScholarBank@NUS Repository. https://doi.org/10.1186/1471-2164-10-125 | Abstract: | Background: Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish. Results: The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern. Conclusion: Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution. © 2009 Yue et al; licensee BioMed Central Ltd. | Source Title: | BMC Genomics | URI: | https://scholarbank.nus.edu.sg/handle/10635/174463 | ISSN: | 14712164 | DOI: | 10.1186/1471-2164-10-125 |
Appears in Collections: | Elements Staff Publications |
Show full item record
Files in This Item:
File | Description | Size | Format | Access Settings | Version | |
---|---|---|---|---|---|---|
10_1186_1471-2164-10-125.pdf | 478.53 kB | Adobe PDF | OPEN | None | View/Download |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.