Determination of species-specific primers for minisatellite variation analysis among and between populations of turkey vultures (Cathartes aura)

• Main Author: Harris, Chad E.
• Other Authors: Vann, Carolyn N.
• Format: Others
• Published: 2011
• Subjects: Turkey vulture > Genetics.
• Online Access: http://cardinalscholar.bsu.edu/handle/handle/186531; http://liblink.bsu.edu/uhtbin/catkey/1138301

What is the genetic relatedness between and among populations of turkey vultures? By determining genetic relatedness, foraging and roosting behaviors of vultures may be better understood. Also as a result of this research, a system of determining genetic relationship will be developed ultimately allowing evolutionary behaviors of vulture populations including altruism and/or group selection to possibly be uncovered. The purpose of this research was to obtain sequence information in order to design species-specific primers for future comparisons of minisatellite variation among and between populations of turkey vultures. Two different methods for DNA isolation from blood were compared for their ability to produce high quantities of amplifiable DNA. The Rapid Method (Lahiri et al., 1993) yielded 5.6 ug of DNA from 500 ul ofblood with a purity ratio [A260/A2S0] of 0.926, while the protocol using IsocodeTM Stixyielded 4.3 ug DNA from 15 ul of blood and had a higher purity ratio of 1.365. Although both methods yielded amplifiable DNA, better amplification was attained using the IsocodeTM Stix, which was used for the rest of the project. The polymerase chain reaction, using RAPD (Random Amplified Polymorphic DNA) primers (Operon Technologies, Alameda, CA), was performed to obtain DNA regions containing minisatellites. Fragments generated by the OPB 08 primer hybridized to a pool of labeled minisatellite core sequences by Southern hybridization. This minisatellitecontaining fragment (800 bp) was excised from a gel and cloned into a plasmid vector (pCR®2.1-TOPO) producing a recombinant plasmid. The recombinant plasmids werereplicated in E. coli, plasmid DNA was isolated, and the cloned fragment was sequenced for determination of the flanking sequences around the minisatellite core. Multiple colonies (pTpvul 1-4) were picked from the cloning/transformation stages but only one brightly hybridizing colony was chosen for sequencing (pTpvul 1). Sequencing and sequencing analysis proved difficult and no minisatellite core sequences could be located. This could be attributed to extensive secondary structure in the DNA sequence or to recombination within the fragment when grown in E. coli. These flanking sequences, thought to be identical at each locus of the minisatellite in a genome, were to be used as species-specific primers in future minisatelhte-PCR DNA fingerprinting. === Department of Biology