AMAZON RIVER DOLPHIN POLYMORPHISM AND POPULATION DIFFERENTIATION OF MHC CLASS II PEPTIDES - Biology, Evolution and Conservation of River Dolphins Within South America and Asia - page 120

M ATERIALS AND M ETHODS
We analyzed pink river dolphin skin biopsies from the main rivers or tributaries of the
three major basins in Northern South America. Twenty-three (23) animals were from the
Beni-Mamoré basin in the Bolivian Amazon (Mamoré, Iruyañez, Iténez-Guaporé and
Ipurupuru rivers). Another 27 were from the Peruvian Amazon (Napo-Curaray, Marañón-
Samiria, Ucayali-Tapiche rivers) as well as from the Colombian Amazon (Putumayo River),
and 10 were from the Orinoco River and some of its tributaries (Guaviare and Inirida rivers)
(Figure 1).
Figure 1. Sample locations and number of animals sampled (and sequenced) in the Beni-Mamoré,
Amazon and Orinoco basins. In the Orinoco basin, three rivers were sampled (Orinoco River, Inírida
River, Guaviare River). In the Amazon basin, five rivers were sampled (Putumayo River, Napo River,
Curaray River, Marañón River, Ucayali River). In the Bolivian Amazon, two rivers were sampled
(Mamoré and Iténez rivers).
Total genomic DNA was extracted using standard organic protocol (Sambroo et al .,
1989) and used to amplify via PCR the second exon of MHC class II DQB using primers
previously proved in other Cetacea (Murray et al . 1995): 5'-CAT GTG CTA CTT CAC CAA
CGG-3' (forward) and 5'-CTG GTA GTT GTG TCT GCA CAC-3' (reverse). The
amplification reactions were conducted on a Techne Genius Thermocycler or in a BioRad
Cycler in a 25 μl total volume using 0.6 μM of each primer, 1X buffer reaction, 3.5 mM
MgCl 2 , 0.4 μM dNTP, 1 U Taq polymerase (Invitrogen) and 25 to 50 ng of DNA. Thermal
cycling conditions comprised an initial denaturation at 95°C for 5 min, followed by 30 cycles
of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 10 min.
PCR products of expected size from the first assayed individual were cloned and sequenced