title: Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones.
abstract: A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol-chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2min instead of 24h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8±0.2°C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40ng/μl and proved to be a superior alternative to the phenol-chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.
authors.affiliation: Department of Bioengineering, Faculty of Engineering, Canakkale Onsekiz Mart University, 17020 Canakkale, Turkey; Novel Fluidic Technologies and Applications Group, Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornova-Izmir, Turkey.
authors.forename: Elif
authors.initials: E
authors.lastname: Ozdemir-Kaynak
authors.affiliation: Novel Fluidic Technologies and Applications Group, Department of Bioengineering, Faculty of Engineering, Ege University, 35100 Bornova-Izmir, Turkey. Electronic address: ozlem.yesil.celiktas@ege.edu.tr.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Yesil-Celiktas
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D002725:Chloroform / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D005269:Femur / Q000737:chemistry*; D006801:Humans; D008872:Microwaves; D010636:Phenols / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
chemical_list: D010636:Phenols; D012822:Silicon Dioxide; D002725:Chloroform; D004247:DNA
keywords: Bones; DNA; Inhibition; Microwave-assisted digestion; Polymerase chain reaction