THE MULTILOCUS DNA FINGERPRINT FOR ANALYSIS OF GENETICS POLYMORPHISM OF GENTOO PENGUINS (PYGOSCELIS PAPUA)

M.V.Dybkov, G.D.Telegeev, A.N.Dubrovskaya

For analysis of minisatellites the DNA of penguins digest (separately) by endonucleases BsuRI, MvaI. After ethanol precipitation, DNA fragments separate in 20 cm 1% agarose gel at 2 V/cm for 36 hour at room temperature in TBE buffer and transfer to ny lon membrane Hybond-N by electrotransfering in 25 mM phosphate buffer (pH 6.5) for 2.5 hour with forced cooling at 5-15^oC. The transferred DNA fragment fixing at membranes by heated for 2 hour at 80^oC at vacuum.
Radioactive probe based on M13 phage obtain on phage M13 DNA by used specific primer, annealing near the hypervariable region of the phage gene III with following synthesize by Klenov fragment, alpha-P³² dCTP and "cold" dNTP. The second probe obtain by tande m ligate of two self-complementary primers (which form tandem repeats analogous of core sequences of 33.15 probe by A.Jeffreys) and clone it at pUC19 vector.
The probe obtains by Klenov fragment with M13 sequences primers. Membranes prehybridizated in buffer G (7% SDS, 1% BSA, 5mM EDTA, 50mM phosphate buffer, pH 6,5) for 18hour at 60^oC.
After this, radioactive probes add to filters and incubate solution for 24 hour at 60^oC. After hybridisation filters wash twice by 5xSSC, 0,1% SDS at room temperature, twice by 3xSSC.
After drying, they autoradiography with X-raying films for 2-14 days.
The size of fragments on the DNA fingerprints determine by comparison with the molecular weight markers - lambda/HindIII, lambda/EcoRI, and M13mp18/ClaI and the control DNA sample. The distance from the start to the centre of a marker band at the fingerprint is me asure. For analysis of bands the fingerprint to divide into discrete segments proceed from accuracy measurement detection the band position + 1 mm and possible errors in determination of the size of fragments with different molecular weights.
Binary matrices make and calculate by software package GELSTATS by S. Pelikan and S.H. Rogstad. Following data obtain at the output: summary stats on band number and frequency, results on similarity (definition of similarity, permutation tests on similarities, mean between group similarities), heterozygosity computations (estimating allele frequenc ies, estimating heterozygosity, proportion loci polymorphic, comparing heterozygosities, Fst according to Nei).

An authors express one's thanks to the INTAS for support of this research (INTAS project 01-0517).

Last Modified