Kvalitativna i kvantitativna analiza humanih bioloških tragova minimalnih graničnih količina u forenzičkim analizama DNK

Abstract

Ćelije kože ostavljene na površini bilo kog predmeta nakon kontakta sa njim mogu biti izvor DNK materijala. Ova vrsta biološkog materijala, u odnosu na količinu DNK koju je moguće izolovati iz nje, obično je mnogo manje izdašna u odnosu na tragove koji se rutinski analiziraju (krv, semena tečnost, pljuvačka). Iako visoko osetljive tehnike analize omogućavaju dobijanje rezultata iz minimalnih količina DNK, još uvek postoje znatne poteškoće u radu sa ovakvim uzorcima, i to iz više razloga. Različit kvalitet i kvantitet izolovane DNK, te posebno izraženi stohastički efekti samo su neki od njih. Zbog toga je proces analize DNK materijala izolovan iz ovakvih uzoraka posebno kompleksan. Biološki uzorci ove vrste se vrlo često nalaze u izuzetno malim količinama (doslovno tragovima), što se postojećim rutinskim metodama analize DNK ne mogu pouzdano interpretirati. U cilju poboljšavanja rezultata analize DNK materijala dobijenog izolovanjem biološkog materijala zaostalog na površini dodirnutih predmeta, analizirano je više od 700 otisaka prstiju. Svaki korak procesa je testiran posebno u cilju dobijanja STR profila koji su bar 70% kompletni, te stoga mogu biti uneti u baze podataka. Kompletnost profila je određivana na osnovu broja dobijenih alela iz 15 testiranih lokusa (najviše moguće 30 alela). Testirani su sledeći koraci u proceduri dobijanja DNK profila: prikupljanje uzoraka, izolovanje DNK, umnožavanje STR lokusa kao i detekcija proizvoda umnožavanja kapilarnom elektroforezom. Ćelije su prikupljene korišćenjem mikrosfera, lepljivih traka, lepila ili pamučnih briseva natopljenih različitim rastvorima. DNK materijal je izolovan korišćenjem interno razvijenih metoda i/ili komercijalno dostupnih kompleta hemikalija za njenu izolaciju. Izolovana DNK je umnožavana Identifiler® kompletom, korišćenjem različitog broja PCR ciklusa...

Skin flakes left on an object after it has been touched or handled could be a source of DNA. These skin flakes tend to be deposited in considerably smaller amounts than from routinely tested cells of blood, semen or saliva. Although, highly sensitive DNA analysis procedures are able to provide results from trace amounts of DNA there are still some fundamental difficulties inherent to these samples, including variability in quality and quantity of extracted DNA and exaggerated stochastic effects, making it hard to reliably interpret DNA profiles of these samples. These types of samples could also carry skin flakes in trace, which currently applied methodology of testing frequently cannot interpret. In order to improve the results from touched DNA samples, over 700 fingerprints were tested. Each step of the workflow for genotyping was assessed with the goal to generate STR profiles that were at least 70% complete and therefore database eligible. The profiles were calculated from the number of obtained alleles with a maximum of 30 for the 15 amplified STR loci. The steps evaluated in the workflow included sample collection, DNA extraction, STR amplification and detection utilizing capillary electrophoresis. Cells were collected using microglobes, tapes, glues, or cotton swabs moistened with different solutions. DNA extraction was assessed with methods designed in the laboratory and commercially available extraction kits. Extracted DNA was amplified with Identifiler® kits using various number of PCR cycles. These comparisons led to the best method that generated a database eligible STR profiles from almost 70% of tested fingerprints. This method suggested collection of fingerprints by swabbing with cotton swab moistened in detergent solution, then extracting DNA using a commercially available extraction kit that uses enzyme activated at a high temperature, followed by amplification at higher PCR cycle number and analysis at longer injection time and higher voltage during capillary electrophoresis...