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BMC Genomics

, 10:125

First Online: 25 March 2009Received: 22 December 2008Accepted: 25 March 2009

Abstract

BackgroundMicrosatellites 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.

ResultsThe 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.

ConclusionNormalization 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.

List of abbreviationscDNAcomplementary DNA

ESTexpressed sequence tag

DSNduplex-specific nuclease.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-10-125 contains supplementary material, which is available to authorized users.

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Autor: Gen Hua Yue - Ze Yuan Zhu - Chun Ming Wang - Jun Hong Xia

Fuente: https://link.springer.com/article/10.1186/1471-2164-10-125



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