By Beth Shapiro, Michael Hofreiter
Examine into historical DNA started greater than 25 years in the past with the book of brief mitochondrial DNA series fragments from the quagga, an extinct relative of the zebra. historic DNA learn particularly won momentum following the discovery of PCR, which allowed thousands of copies to be made up of the few final DNA molecules preserved in fossils and museum specimens. In historical DNA: equipment and Protocols professional researchers within the box describe some of the protocols which are now typical to check historical DNA. those comprise directions for developing an old DNA laboratory, extraction protocols for a variety of diverse substrates, information of laboratory ideas together with PCR and NGS library practise, and proposals for applicable analytical ways to make feel of the sequences bought. Written within the hugely profitable tools in Molecular Biology™ series layout, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and key pointers on troubleshooting and fending off identified pitfalls. Authoritative and sensible, old DNA: equipment and Protocols seeks to assist scientists within the additional research of historic DNA and the methodological ways in historic examine.
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Additional resources for Ancient DNA (Methods in Molecular Biology, v840)
A recent reanalysis of the available mtDNA data (1,448 base pairs (bp) of the 12S rRNA and cytochrome b gene) with increased taxonomic coverage and an additional 169 bp of ATP8 (also from the mitochondrion) suggested a close but not highly supported evolutionary relationship with the New World pigeons, Patagioenas (7). As mtDNA and nuclear DNA phylogenies are not always congruent, we targeted a nuclear intron to further examine passenger pigeon phylogeny. Analyses of these new data confirmed the sister relationship between the passenger pigeon and New World pigeons (Patagioenas) and provided moderately strong support for the Ectopistes–Patagioenas clade (1).
5. Low retention or siliconized tubes are recommended, which reduce DNA loss due to tube wall effects. 6. The silica suspension is stable for at least 1 month. 7. Do not exceed 250 mg/5 mL extraction buffer. It is possible to proportionally scale the extraction up or down when more or less sample material is used; use 1 mL/50 mg. It is crucial to adjust the binding buffer volume accordingly (see Notes 10 and 11). 8. Incubation can also be performed at 37°C, where proteinase K is more active than it is at room temperature.
3. Centrifuge for 5 min to separate the layers. The speed of centrifugation will depend on the volume of the digestion mix, the centrifuge capacity, and the maximum speed designation of the tubes being used. It is generally advisable to use the maximum speed possible. If after 5 min the layers have not fully separated, extend the centrifugation time. 4. Carefully remove the upper aqueous layer. Be careful not to remove the protein-containing interface. Discard the lower, phenol layer (see Note 8).
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