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RichardE.Green,JohannesKrause,SusanE.Ptak,AdrianW.Briggs,MichaelT.Ronan,JanF.Simons,LeiDu, MichaelEgholm,JonathanM.Rothberg,MajaPaunovic&SvantePbo
NatureVol444|16November2006|doi:10.1038/nature05336
BrbaraVerduzco|JHU410.666GenomicSequencingandAnalysis|September2012
Introduction
Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. Neanderthals were first recognized as a distinct group of hominids from fossil remains discovered 150 years ago at Feldhofer in Neander Valley, outside Dsseldorf, Germany.
A Neanderthal nuclear genome sequence would be an invaluable resource for annotating the human genome to mark when remarkable morphological, behaviorural, and cognitive changes occurred.
Methodology
IdentificationofaNeanderthalfossilforDNAsequencing Avoid: Degradation Chemicaldamage DNAisirretrievableormisread Contamination specimens,labreagents,andinstruments
mtDNA
Hypervariable region
Picksample
PCR
Clone
454
BFX Analysis
More PCRs
More Analysis
Check for biomolecular preservation by using samples of a few milligrams for amino acid analysis. The vast majority of these samples had low overall contents of amino acids and/or high levels of amino acid racemization, a stereoisomeric structural change that affects amino acids in fossils, indicating that they are unlikely to contain retrievable endogenous DNA.
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AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336
Preparation of sample
PCR
Figure1 Ratio of Neanderthal to modern human mtDNA in six hominid fossils. For each fossil, primer pairs that amplify a long (119 base pairs; upper lighter bars) and hort (63 base pairs; lower darker bars) product were used to amplify segments of the mtDNA hypervariable region. The products were sequenced and determined to be either of Neanderthal (yellow) or modern human (blue) type.
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AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336
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AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336
Overview of Results
Figure4 LocationonthehumankaryotypeofNeanderthalDNAsequences
AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336
Overview of Results
Figure 5 Schematic tree illustrating the number of nucleotide changes inferred to have occurred on hominoid lineages.
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AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336
Bridgeamplification Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.
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AnalysisofonemillionbasepairsofNeanderthalDNA|NatureVol444|16November2006|doi:10.1038/nature05336
PresentationGuidelines:
Timeforpresentationwillbeapproximately20 minutesperstudent Eachstudentwillpresentasummaryofthe paperconsistingofthefollowing:
Principlesofthemethod(1slide) Preparationofsample(1slide) Sequencingprocedureandmethod(2slides) Overviewofresults(2slides) Comparebrieflytoothermethods(1slide)
Gettowork!
Afterthepresentation,emailtheinstructors yourslides!
10 BrbaraVerduzco|JHU410.666GenomicSequencingandAnalysis|September2012