Fishing for ancient DNA (original) (raw)
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Chapter 2: A Phenol-Chloroform Protocol for Extracting DNA from Ancient Samples
"Research into ancient DNA began more than 25 years ago with the publication of short mitochondrial DNA sequence fragments from the quagga, an extinct relative of the zebra. Ancient DNA research really gained momentum following the invention of PCR, which allowed millions of copies to be made of the few remaining DNA molecules preserved in fossils and museum specimens. In Ancient DNA: Methods and Protocols expert researchers in the field describe many of the protocols that are now commonly used to study ancient DNA. These include instructions for setting up an ancient DNA laboratory, extraction protocols for a wide range of different substrates, details of laboratory techniques including PCR and NGS library preparation, and suggestions for appropriate analytical approaches to make sense of the sequences obtained. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Ancient DNA: Methods and Protocols seeks to aid scientists in the further study of ancient DNA and the methodological approaches in ancient research. Content Level » Professional/practitioner Keywords » NGS - PCR - Pleistocene-age DNA - ancient DNA - ancient bone - bone hydroxyl-apatite - hydroxyl-apatite crystals - mitochondrial genomes - phylogeographic patterns - soft tissue "
Development and Optimization of a Silica Column-Based Extraction Protocol for Ancient DNA
Genes
Rapid and cost-effective retrieval of endogenous DNA from ancient specimens remains a limiting factor in palaeogenomic research. Many methods have been developed to increase ancient DNA yield, but modifications to existing protocols are often based on personal experience rather than systematic testing. Here, we present a new silica column-based extraction protocol, where optimizations were tested in controlled experiments. Using relatively well-preserved permafrost samples, we tested the efficiency of pretreatment of bone and tooth powder with a bleach wash and a predigestion step. We also tested the recovery efficiency of MinElute and QIAquick columns, as well as Vivaspin columns with two molecular weight cut-off values. Finally, we tested the effect of uracil-treatment with two different USER enzyme concentrations. We find that neither bleach wash combined with a predigestion step, nor predigestion by itself, significantly increased sequencing efficiency. Initial results, however,...
Advances in ancient DNA studies
2006
The first aDNA studies used bacterial cloning to amplify small sequences retrieved from skins of animal and human mummies, and revealed the inefficient reaction kinetics of this technique (Higuchi et al., 1984; Paabo 1985, 1989). These studies demonstrated that the genetic material surviving in ancient specimens was often principally microbial or fungal in origin. Endogenous DNA was generally limited to very low concentrations of short, damaged fragments of multi-copy loci such as mitochondrial DNA (mtDNA). The invention of the polymerase chain reaction (PCR) made it possible to routinely amplify and study even single surviving molecules, allowing the number and range of aDNA studies to diversify rapidly (Paabo 1989; Paabo & Wilson 1988; Paabo et al., 1989; Thomas, 1989). However, the enormous amplifying power of PCR also means that contamination from modern DNA becomes a major problem. Contamination is especially likely when previously amplified PCR products are present. False posi...
Ancient DNA Extraction from Skeletal Material v1
protocols.io, 2019
Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from skeletal material powder (e.g. bone, teeth), modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110).
ANCIENT DNA RESEARCH: ONGOING CHALLENGES AND CONTRIBUTION TO MEDICAL SCIENCES
Life gave rise on our planet 3-4 billion years ago and since then, living organisms (from one cell to multicellular organisms) have undergone many genetic, phenotypic and communal changes. Scientists have been able to shed light on only a small part of this evolutionary process, but with the development of new techniques our knowledge is expanding day by day. For the past 30 years ancient DNA studies have aided us in understanding the molecular basis of the changes observed in living organisms. Ancient DNA (aDNA) is the genetic material obtained from biological remains (bones, teeth, plant seeds, etc.) acquired from archaeological and paleontological excavations. In the present review, molecular studies carried out to date, contributions of ancient DNA to medical sciences, as well as basic problems encountered in obtaining and using aDNA have been discussed.
BioTechniques, 2021
DNA extraction is of utmost importance in archaeobiology, as it determines the success of further DNA analyses. This study concentrates on the success of ancient DNA extraction using silica spin columns and PCR-based analysis from archaeological skeletal material and investigates the influence of sample quantity, lysis time and lysis temperature during sample preparation. The results show that lysis times ranging from 2 to 48 h are suitable, and that lysis should be carried out at a constant temperature of 56°C. Concerning sample quantity, 10 mg for mitochondrial DNA and 50 mg for chromosomal DNA are sufficient for high quality analyses. Thus invaluable sample material can be saved, and time of sample preparation can be reduced considerably.