http___medlib.mef.hr_858__1.xml
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Mitrečić, Dinko and Mavrić, Sandra and Vrabec Branica, Božica and Gajović, Srećko (2008) Mice genotyping using buccal swab samples: an improved method. Biochemical Genetics, 46 (3-4). Abstract Routine methods used to genotype mice involve isolation of DNA from partially amputated neonate's tail, toe, or ear. The inevitable drawbacks of such techniques are the animal's pain response and the increased time and funds required for DNA purification. In order to implement a noninvasive and simple protocol for mouse DNA isolation, we have improved the method based on samples collected by swabbing of the inner cheek. Combining alkaline and temperature lysis, it was possible to isolate a DNA solution ready for PCR in less than an hour. Testing the method on three different mouse lines showed that it is highly efficient, the volume of the PCR samples could be reduced to 25 microl, and fragments up to 800 bp were successfully amplified. This protocol reduces animal discomfort, shortens the time for DNA isolation, and enables amplification of larger DNA fragments with optimal success rate, thus considerably facilitating large-scale genotyping of different mouse lines. Actions (login required) Downloads Downloads per month over past year
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Vrabec Branica, Božica (2010) Primjena lužnate lize u izolaciji DNA kao novi pristup analizi genskih biljega bronhalne sluznice iz rutinskih citoloških uzoraka dobivenih tijekom bronhoskopije. PhD thesis, Sveučilište u Zagrebu. Croatian abstract Tek je mali broj radova objavljen o izolaciji DNA iz rutinskih citoloških uzoraka. Neupitan nedostatak većine metoda izolacije DNA je u složenosti postupka koji uključuje obvezatno dodatno pročišćavanje DNA. U ovom radu uspostavljen je inovativni jednostavni protokol za izolaciju humane DNA iz rutinskih citoloških uzoraka vezanih za karcinom pluća kao što je aspirat bronha, te iz briseava bukalne i vjeđne sluznice. Pokazali smo da je kombinacijom lužnate i temperaturne lize moguće izolirati DNA adekvatnu za metodu lančane reakcije polimeraze (PCR) u manje od sat vremena. Testiranje metode amplifikacijom fragmenata spolnih kromosoma pokazalo je da je metoda efikasna i pouzdana. Prezentirani protokol omogućuje očuvanje visoko kvalitetene DNA prikladne za dijagnostičke postupke koji se osnivaju na lančanoj reakciji polimerazom. Osim dobro poznate uloge u razvoju karcinoma cerviksa, postoje naznake da bi HPV mogao imati ulogu i u kancerogenezi karcinoma pluća. U rutinskim citološkim uzorcima aspirata bronha 84 pacijenata s karcinomom pluća izolirana je DNA te se utvrđivala prisutnost visoko rizičnih tipova humanog papiloma virusa tipa 16, 18 i 33 metodom lančane reakcije polimeraze. Rezultate smo uspoređivali s onima iz uzoraka briseva bukalne i vjeđne sluznice istih pacijenata. Samo su tri pacijenta bila HPV pozitivna u uzorcima aspirata bronha: jedan je bio HPV tip 16 pozitivan, jedan HPV tip 18, a jedan HPV tip 33 pozitivan. Pacijenti pozitivni na tip 18 i 33 HPV-a bili su pozitivni na iste tipove virusa i u uzorcima brisa bukalne sluznice. Svih 84 pacijenata bilo je HPV negativno u uzorcima briseva vjeđne sluznice. Naši rezultati svjedoče o niskoj prevalenciji HPV-a u pacijenata s karcinomom pluća u Hrvatskoj, što se podudara s rezultatima nekih drugih europskih zemalja i govori u prilog tome da je vjerojatnije da HPV nema veću ulogu u razvoju karcinoma pluća ove zemljopisne regije. English abstract There are only a few systematic reports about DNA extraction from routine diagnostic cytological specimens. An inevitable drawback of such techiniques is obligatory DNA purification. In our study, a simple protocol is implemented for human DNA isolation from cytological specimens related to lung cancer, like bronchial aspirates and samples collected by swabbing of the inner cheek and eyelid. By combining alkaline and temperature lyses it was possible to isolate DNA solution ready for PCR in less than an hour. Testing the method used for amplification of sex chromatin gene fragments showed that it is highly efficient and reliable. The presented protocol preserves high-quality DNA that is suitable for diagnostic procedures based on PCR methods. Besides its well-known role in cervical carcinoma, HPV is also suggested to be involved in lung cancer development. We have used routine bronchial aspirates from 84 patients with lung carcinoma for DNA extraction and then performed polymerase chain reaction for high-risk HPV types 16, 18 and 33. The results were compared to those obtained from buccal and eyelid mucosa. Only three patients were positive for HPV in bronchial aspirates: one for HPV 16 type, one for HPV 18 type, and one for HPV 33. Patients HPV 18 and HPV 33 positive had also HPV positive sample of buccal mucosa. All 84 patients were HPV negative in eyelid mucosa samples. Our data indicated the low prevalence of HPV in patients with lung carcinomas in Croatia, similar to some other European countries, therefore it seems unlikely that HPV contributes to the development of lung carcinomas in this region. Actions (login required)