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A simple and efficient method for isolating polymorphic microsatellites from cDNA.

Yue GH, Zhu ZY, Wang CM, Xia JH - BMC Genomics (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Population Genetics Group, Temasek Life Sciences Lab, 1 Research Link, National University of Singapore, 117604 Singapore. genhua@tll.org.sg

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 degrees 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.

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Schematic presentation of the method for microsatellite enrichment from normalized cDNA. Details of each step can be found in the section "Methods".
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Figure 1: Schematic presentation of the method for microsatellite enrichment from normalized cDNA. Details of each step can be found in the section "Methods".

Mentions: In order to increase the efficiency of enrichment of microsatellites, we tried to reduce the redundancy of cDNA by normalizing cDNA using duplex-specific nuclease (DSN) [20] before enrichment of CA- and GA-microsatellites (Figure 1). After cDNA normalization, redundant cDNA were removed (Figure 2). Two normalized cDNA libraries, one enriched for CA-microsatellites and another for GA-repeats were created. From each library, 192 clones were sequenced in both ends respectively. Eighty-eight (45.8%) and 51 (26.5%) clones of the 192 clones from the normalized cDNA libraries enriched for CA- and GA-repeats respectively, contained microsatellites (Table 1). The redundancy of clones was substantially reduced. In the 88 clones containing microsatellites from the cDNA library enriched for CA-microsatellites, 41 were singletons, the remaining 47 were included in 10 contigs. A total of 51 (26.5%) unique microsatellites were obtained from 192 sequenced clones. In the 51 clones containing microsatellites from the cDNA library enriched for GA-repeats, 35 were singletons, and 16 were included in 5 clusters. A total of 40 (20.8%) unique microsatellites we obtained from 192 sequenced clones (Table 1). In comparison to the efficiency of microsatellite enrichment from unnormalized cDNA, the efficiency was about three folds increased by using normalized cDNA (for CA enrichment: 26.5% vs. 9.9%; for GA enrichment: 20.8% vs. 7.3%). Therefore, decreasing the prevalence of clones representing abundant transcripts before microsatellite enrichment by normalization of cDNA is essential for microsatellite isolation from cDNA. The normalization of cDNA using DSN was very simple and highly efficient in comparison to other cDNA normalization methods [21]. The whole procedure of microsatellite enrichment starting from normalization of cDNA lasted only 5 days. Application of this method to isolate microsatellites from cDNA of grass carp brain got similar results (data no shown). Therefore the method is robust and reproducible.


A simple and efficient method for isolating polymorphic microsatellites from cDNA.

Yue GH, Zhu ZY, Wang CM, Xia JH - BMC Genomics (2009)

Schematic presentation of the method for microsatellite enrichment from normalized cDNA. Details of each step can be found in the section "Methods".
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667190&req=5

Figure 1: Schematic presentation of the method for microsatellite enrichment from normalized cDNA. Details of each step can be found in the section "Methods".
Mentions: In order to increase the efficiency of enrichment of microsatellites, we tried to reduce the redundancy of cDNA by normalizing cDNA using duplex-specific nuclease (DSN) [20] before enrichment of CA- and GA-microsatellites (Figure 1). After cDNA normalization, redundant cDNA were removed (Figure 2). Two normalized cDNA libraries, one enriched for CA-microsatellites and another for GA-repeats were created. From each library, 192 clones were sequenced in both ends respectively. Eighty-eight (45.8%) and 51 (26.5%) clones of the 192 clones from the normalized cDNA libraries enriched for CA- and GA-repeats respectively, contained microsatellites (Table 1). The redundancy of clones was substantially reduced. In the 88 clones containing microsatellites from the cDNA library enriched for CA-microsatellites, 41 were singletons, the remaining 47 were included in 10 contigs. A total of 51 (26.5%) unique microsatellites were obtained from 192 sequenced clones. In the 51 clones containing microsatellites from the cDNA library enriched for GA-repeats, 35 were singletons, and 16 were included in 5 clusters. A total of 40 (20.8%) unique microsatellites we obtained from 192 sequenced clones (Table 1). In comparison to the efficiency of microsatellite enrichment from unnormalized cDNA, the efficiency was about three folds increased by using normalized cDNA (for CA enrichment: 26.5% vs. 9.9%; for GA enrichment: 20.8% vs. 7.3%). Therefore, decreasing the prevalence of clones representing abundant transcripts before microsatellite enrichment by normalization of cDNA is essential for microsatellite isolation from cDNA. The normalization of cDNA using DSN was very simple and highly efficient in comparison to other cDNA normalization methods [21]. The whole procedure of microsatellite enrichment starting from normalization of cDNA lasted only 5 days. Application of this method to isolate microsatellites from cDNA of grass carp brain got similar results (data no shown). Therefore the method is robust and reproducible.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Population Genetics Group, Temasek Life Sciences Lab, 1 Research Link, National University of Singapore, 117604 Singapore. genhua@tll.org.sg

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 degrees 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.

Show MeSH