NDLI: Identification of New World monkey MHC-DRB alleles using PCR, DGGE and direct sequencing

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Identification of New World monkey MHC-DRB alleles using PCR, DGGE and direct sequencing

Content Provider	Springer Nature Link
Author	Middleton, Simon A. Anzenberger, Gustl Knapp, Leslie A.
Copyright Year	2004
Abstract	Identification of New World monkey MHC-DRB alleles has previously relied upon labor-intensive cloning and sequencing techniques. Here we describe a rapid and unambiguous way to distinguish DRB alleles in New World monkeys using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of New World monkey DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. The validity of this typing procedure was confirmed by the identification of all DRB alleles previously characterized by cloning and sequencing techniques from an individual cotton-top tamarin. Importantly, our analysis revealed DRB alleles not previously identified in this reference animal. Following validation of our technique, the protocol was employed for the characterization of MHC-DRB alleles in four other species of New World monkey: the pygmy marmoset, white-faced saki monkey, long-haired spider monkey and owl monkey. Using this technique, we identified five alleles from the cotton-top tamarin, five alleles from the owl monkey, three alleles from the long-haired spider monkey, three alleles from the white-faced saki monkey and two alleles from the pygmy marmoset. On the basis of phylogenetic tree analyses, 13 new DRB alleles were assigned to eight different MHC-DRB lineages. Whereas traditional DRB typing via cloning and sequencing provides limited information, our new technique provides a simple and relatively rapid way of identifying New World monkey MHC-DRB alleles.
Starting Page	785
Ending Page	790
Page Count	6
File Format	PDF
ISSN	00937711
Journal	Immunogenetics
Volume Number	55
Issue Number	11
e-ISSN	14321211
Language	English
Publisher	Springer-Verlag
Publisher Date	2004-01-09
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	New World monkey Cotton-top tamarin MHC-DRB Denaturing gradient gel electrophoresis Direct sequencing
Content Type	Text
Resource Type	Article
Subject	Genetics Immunology

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