On first stage some decamer primers (kits OPE-OPI, Operon Technologies,
USA) screen. For this prepare PCR-mix in volume of 30 ml containing 50 ng of
genomic DNA, 1xPCR buffer (100mM tris-HCl, 60mM (NH4)
2SO4, 0,08% Twin-20 pH8,8),
30 pmol primer, 0,2 mM each of dNTP and 1 U of Taq DNA polymerase.
Amplification at 40 cycles for 1 min. at 94oC, for 1 min at 36oC and 2 min
at 72oC by used the fastest available transitions bet ween each temperature.
Amplified products analyse by electrophoresis agarose gel and detect by
staining with ethidium bromide. A negative control without any DNA template
and positive control with one probe of DNA penguins include in each
amplification batch.
The primers with a high polymorphism use for analyse
genetic similarity and diversity within and between populations by RAPD-PCR
and bandsharing analysis. For this, after optimisation of RAPD reaction
conditions for selected primers, to analyse all collected probes of blood of
penguins.
Each band consider as the RAPD marker. All the amplification
reaction repeat two times for analysis and only reproducible bands to
consider.
The RAPD data use for comparison within- and between-populations
variations. For this make binary matrices and calculate main characteristic -
summary stats on band number and frequency, Jaccard's similarity coefficient,
Nei and Li si milarity coefficient and construction UPGMA dendrogram.
An authors express one's thanks to the INTAS for support of this research (INTAS project 01-0517).
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