DNA-Analytik. Dr. Holger Klapproth

Transkript

1 DNA-Analytik Dr. Holger Klapproth

2 International Consortium Completes Human Genome Project All Goals Achieved; New Vision for Genome Research Unveiled BETHESDA, Md., April 14, 2003 The International Human Genome Sequencing Consortium, led in the United States by the National Human Genome Research Institute (NHGRI) and the Department of Energy (DE), today announced the successful completion of the Human Genome Project more than two years ahead of schedule. Also today, NHGRI unveiled its bold new vision for the future of genome research, officially ushering in the era of the genome. The vision will be published in the April 24 issue of the journal Nature, coinciding with the 50th anniversary of Nature s publication of the landmark paper by Nobel laureates James Watson and Francis Crick that described DNA s double helix. Dr. Watson also was the first leader of the Human Genome Project. The international effort to sequence the 3 billion DNA letters in the human genome is considered by many to be one of the most ambitious scientific undertakings of all time, even compared to splitting the atom or going to the moon.

3 7. Vorlesungsstunde: Von der Restriktionskarte zu HUG Der Weg zur Sequenz des Genoms? Guanin H 2 N HN N N N H P H P H P H 5 H 2

4 Genkarten Cytogenetische Genkarten relative Einheit aufgrund der Rekombintionshäufigkeit Einheit: Morgan (Centimorgan) Physikalische Karten Restriktionsanalyse Genkarten Einheit: Basenpaare (bp) bzw. Kilobasen

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6 Restriction Enzymes: Microscopic Scalpels Isolated from various bacteria, restriction enzymes recognize short DNA sequences and cut the DNA molecules at those specific sites. (A natural biological function of these enzymes is to protect bacteria by attacking viral and other foreign DNA.) Some restriction enzymes (rare-cutters) cut the DNA very infrequently, generating a small number of very large fragments (several thousand to a million bp). Most enzymes cut DNA more frequently, thus generating a large number of small fragments (less than a hundred to more than a thousand bp). n average, restriction enzymes with 4-base recognition sites will yield pieces 256 bases long, 6-base recognition sites will yield pieces 4000 bases long, and 8-base recognition sites will yield pieces 64,000 bases long. Since hundreds of different restriction enzymes have been characterized, DNA can be cut into many different small fragments.

7 Erstellen einer Restriktionsmappe Schneiden der DNA durch zwei oder mehr verschiedene Restriktionsenzyme Klonieren der Fragmente Analyse der Überlappungsbereiche der Fragmente EcoRI Hind III

8 What is the Human Genome Project? U.S. govt. project coordinated by the Department of Energy and the National Institutes of Health goals ( ) identify the approximate 100,000 genes in human DNA determine the sequences of the 3 billion bases that make up human DNA store this information in databases develop tools for data analysis address the ethical, legal, and social issues that arise from genome research

9 Doch nur Wie viele Gene hat der Mensch? Die neuste und genauste Schätzung vom ktober 2004 geht davon aus, dass der Mensch nur etwa 20'000 bis 25'000 Gene hat! Das sind nicht viel mehr als bei einem einfachen Fadenwurm zu finden sind! In den 80er Jahren waren die Forscher noch überzeugt, dass der Mensch oder noch mehr Gene haben müsste. Nach der Entzifferung des menschlichen Genoms im Jahre 2001 lauteten die ersten Schätzungen noch auf 30'000-40'000. Nun rätseln die Wissenschafter, wie so wenig Gene einen so komplexen rganismus wie den Menschen zu steuern vermögen.

10 Genomgrößen rganismus Genomgröße1 Gene Gendichte2 λ-phage Blattfloh-Endosymbiont Carsonella ruddii 1, Darmbakterium Escherichia coli 4, Bäckerhefe Saccharomyces cerevisiae Trichoplax adhaerens Fadenwurm Caenorhabditis elegans Acker-Schmalwand Arabidopsis thaliana Taufliege Drosophila melanogaster Kugelfisch Fugu rubripes 3, Kohl Brassica oleracea 5,99-8, Mensch Homo sapiens 3, ca Teichmolch Triturus vulgaris 2, in Basenpaaren 2Anzahl der Gene pro Millionen Basenpaare Quelle: Wikipedia

11 Anteil an nicht-genen im Genom Quelle: Wikipedia

12 Whose genome is being sequenced? the first reference genome is a composite genome from several different people generated from primary samples taken from numerous anonymous donors across racial and ethnic groups

13 Separating Chromosomes Flow sorting Pioneered at Los Alamos National Laboratory (LANL), flow sorting employs flow cytometry to separate, according to size, chromosomes isolated from cells during cell division when they are condensed and stable. As the chromosomes flow singly past a laser beam, they are differentiated by analyzing the amount of DNA present, and individual chromosomes are directed to specific collection tubes. Somatic cell hybridization In somatic cell hybridization, human cells and rodent tumor cells are fused (hybridized) over time, after the chromosomes mix, human chromosomes are preferentially lost from the hybrid cell until only one or a few remain. Those individual hybrid cells are then propagated and maintained as cell lines containing specific human chromo -somes. Improvements to this technique have generated a number of hybrid cell lines, each with a specific single human chromosome.

14 Clonieren eines Chromosoms Chromosom isolieren Chromosom schneiden und in YACs clonieren Yeast Atificial Chromosomes (Megabasen) YACs als Cosmide subklonieren (Kilobasen) Cosmide werden in Baktieren vermehrt, C. sind abgewandelte Genome von Baktriophagen Subklonieren in kurze Fragmente

15 Fishing for Genes Fluorescence In Situ Hybrisisation

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18 Sequenzierungsstrategien Shot Gun Sequencing ungerichtete Sequenzierung der Clone: es müssen überlappende Sequenzen erstellt werden Primer walking: es wird vom Ende her sequenziert, d.h gerichtete Sequenzierung, weniger Sequenzieraufwand

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20 Scientists complete first chapter of book of life with decoding of chromosome 22 An international team of researchers has achieved a scientific milestone by unraveling for the first time the genetic code of an entire human chromosome. Reported in this week's issue of Nature (Dec. 2), researchers at the Sanger Centre near Cambridge, England; University of klahoma, Norman, K; Washington University, St. Louis, M; and Keio University in Japan have succeeded in deciphering the sequence of the 33.5 million "letters," or chemical components, that make up the DNA of chromosome 22. This sequence includes the longest, continuous stretch of DNA ever deciphered and assembled. It is over 23 millionletters in length. 545 Gene identifiziert weiter 200 bis 300 mögliche Gene Gene zwischen 1000 und Basen lang, im Durchschnitt Basen 39% des Chromosoms wird in RNA übersetzt, aber nur 3% codieren für Proteine

21 Endlich: die Sequenz

22 Terminatornukleotide HN CH 3 H P H P H P H 5 N 2 ddttp