Biology Reference
In-Depth Information
preserved in 70% ethanol. DNA was extracted with a standard phenol-chloroform protocol
(Sambrook et al., 2001) or DNeasy
DNA extraction kit (Qiagen®).
Figure 1. Map showing the two sampled areas in Brazil: the Tefé River and Lake and the Mamirauá
Reserve located between the Japurá and Solimões (Amazon) rivers in the central part of the Amazon
basin.
DNA
Amplification
and Sequencing
The mtDNA control region (HVSI) was amplified via the polymerase chain reaction
(PCR) using primers modified from Kocher et al. (1989): L15926 5'
TCAAAGCTTACACCAGTCTTGTAAAACC 3' and H16498 5'
CCTGAAGTAGGAACCAGATG 3'. Each PCR mix contained 20 ng of genomic DNA, 1X
Taq reaction buffer 1B (Phoneutria
- 1.5 mM MgCl
2,
10 mM Tris-HCl (pH 8.4), 50 mM
KCl, 0.1% Triton X-100), 200 μM dNTPs, 0.5 μM of each primer, and 1 unit of Taq DNA
polymerase (Phoneutria
). The PCR cycling was performed in an Eppendorff Gradient
thermocycler at a temperature of 94
o
C for 5 min, followed by 35 cycles at 47
o
C for 45 s, 72
o
C
for 1 min and 94
o
C for 30 s, and a final extension period at 72
o
C for 10 min.
The complete mtDNA Cytochrome
b
(Cyt-b) gene (1140 bp) was amplified using primers
L14121, H15318 and XL14733 (Redondo et al., 2008) and MVZ4 (Kocher et al., 1998). The
cycling profile used for this gene was the same as for the control region, but using 52º C as
the annealing temperature.
PCR products were purified using a 1:1 volume ratio of 10 units/μl of Exonuclease I and
1 unit/μl Shrimp Alkaline Phosphatase (GE Healthcare®) at the temperature of 37
o
C for 45
min and 80
o
C for 15 min. Both strands were sequenced using one of the primers described
above and the DYEnamic ET
dye terminator kit (GE Healthcare®) following the
temperature cycling profile: 94
o
C for 1 min, 35 cycles of 94
o
C at 30 s, annealing at 55
o
C for
Search WWH ::
Custom Search