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Isolation of microsatellites from Catharanthus roseus (L.) G. Don using enriched libraries

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Title Isolation of microsatellites from Catharanthus roseus (L.) G. Don using enriched libraries
 
Creator Bhatia, Sabhyata
Shokeen, Bhumika
 
Subject STMS
Microsatellites
Biotinylated
Streptavidin
C. roseus
Enriched library
Degenerate primers
 
Description Catharanthus roseus is an indispensable source of the anticancerous alkaloids-vincristine and vinblastine, even
though they are produced in trace amounts in vivo. In order to increase the yield of alkaloids, in vitro tissue culture
studies are carried out which result in a large number of lines/cultures. For identification and characterization
of the in vitro cultures, microsatellites in the form of STMS (Sequenced Tagged Microsatellite Sites) markers
are used for identification of genetic polymorphism. STMS markers are also used for assessment of genetic
diversity within natural populations as well as for construction of genetic linkage maps. Isolation of microsatellites
and development of STMS markers typically involves library construction and screening, DNA sequencing,
polymerase chain reaction (PCR) primer design, and PCR optimization. This chapter details two approaches for
the isolation of microsatellite loci. The first approach is based on PCR using microsatellite containing primers
which also have degenerate bases at the 5¢-end that act as anchors preventing the primers from slippage to
the 3¢-end and the subsequent loss of polymorphism. The multi-locus PCR amplified product is cloned and
sequenced. Though this method generates a large number of microsatellites, the major drawback is the high
redundancy observed in this method. The second approach described in this chapter is based on the construction
of a microsatellite enriched library which involves preferential cloning of the microsatellite enriched fraction of
genomic DNA. This method therefore necessitates the isolation of microsatellites through hybridization with
biotin labeled oligoprobe followed by their capture with streptavidin-coated magnetic beads. In comparison to
the first approach, this approach yields less redundant clones with high microsatellite enrichment. Moreover
enriched libraries are 40–60 times more efficient than the conventional small insert genomic libraries.
 
Date 2013-11-18T06:54:07Z
2013-11-18T06:54:07Z
2009
August 2009
 
Type Article
 
Identifier Methods Mol. Biol., 547: 289-302
http://hdl.handle.net/123456789/106
 
Language en
 
Publisher Springer