VL1 Klonierung Flashcards
Definition: Klone
Genetische identische Organismen oder Molekuele, die auf einem gemeinsamen Vorfahren breuhen.
Varianten von Klonierung
Artifizielle Klonierung / Natuerliche Klonierung
Varianten von artifizielle Klonierung
- Reproduktives Klonieren – Das gesamte Tier wird ausgehend von einer Zelle durch asexuelle Reproduktion erstellt
- Therapeutisches Klonieren – Kein identisches Individuum, sondern Erstellung von Gewebe.
- Genklonierung - Gene oder Genfragmente werden kloniert
Varianten von natuerliche Klonierung
- Klonieren von einzelligen Organismen durch Isolierung
- Pflanzliche asexuelle Replikation (Apomixis)
- Zwillinge
Klonierung aus Einzelzellen in Pflanzen: Beispiel / Schritten?
(= tissue culture, micropropagation)
example: carrots
- Plug of tissue is removed, dissociated into single cells (eg from carrot root)
- single cells are transferred to growth medium
- cells divide to form callus
- each callus is transferred to new medium to induce root and shoot formation
- plants are transferred to soil and grown into carrots
result: genetically identical
Klonierung aus Einzelzellen in Pflanzen: Ergebnis?
Genetically identical, or cloned carrots, derived from a single ancestor
Klonierung von Tieren: Ansaetze?
erstes erfolgreiche Experimente an Saugern in den 80ern
zwei Ansaetze:
- embryo splitting
- Kerntransfer
Klonierung von Tieren: Embryo Splitting
Schritte?
- Embryo is split to form two half-embryos
- Embryos are transferred to an unrelated surrogate mother
- Pregnancy is monitored by ultrasound
- sheep gives birth to identical twins
Klonierung von Tieren: Kerntransfer
–
Dolly
–
schritte usw
Dolly - Klonierungseffizienz
– Zellen wurden von einem 6 Jahre alten Finnish Dorset Schaf entnommen und im Labor kultiviert
– 277 dieser Zellen wurden mit 277 Eizellen (ohne Nukleus) fusioniert
– 29 Eizellen überlebten und wurden in Blackface Leihmütter implantiert
– 1 Geburt = Dolly
– Erfolgsrate:
• 0.3% bei Beginn
• 3.4% nach Implantierung
• Nach Befruchtung in utero: 33-50% entwickeln sich
Andere klonierte tiere
goats, cats, cows, dogs
Probleme bei Klonen durch Kerntransfer
- Geringe Lebenserwartung / Krankheiten
- Unterschiede wegen somatischer Mutationen
- Niedrige Erfolgsrate: 0.1 - 3%
- Dedifferenzierung nicht komplett (bei somatischer Zellen (differenziert) Chromatin anders - tabula rasa rueckgaengig zu machen - funktioniert nicht vollstaendig)
- Telomere können verkürzt sein
- Mitochondrien können defekt sein
English three-parent babies:
How?
Purpose?
Produced from genetic material of one man and two women through use of assisted reproductive technologies: mitochondrial manipulation (or replacement) technologies and three-person in vitro fertilization (IVF).
Reproductive technologies used focus on replacing / reducing effects of mutations that occur in mitochondrial DNA.
The various approaches could help women to overcome infertility and could prevent transmission to offspring of potentially debilitating mitochondrial diseases.
Klonierung von Genen
Ziel: Vereinzeln, um zu manipulieren und zu verfielfältigen.
Gen –> Klonierungsvektor –> Wirt –> Vervielfaeltigung
Restriktionsenzyme:
Beispiel?
Funktionsweise?
EcoRI
- binds to the recognition sequence and cuts the DNA into fragments
- EcoRI has sticky ends (as opposed to blunt end)
Restriktionsenzyme?
(=restriction endonuclease, restrictase)
cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites.
One class of broader endonuclease group of enzymes.
Restriktionsenzyme:
Classification and types?
Commonly classified into 5 types: differ in structure and whether they cut their DNA substrate at recognition site, or if recognition and cleavage sites are separate.
More than 3,600 restriction endonucleases known,
Restriktionsenzyme:
Origin? Purpose?
Restriction modification System?
Found in bacteria and archaea.
Provide defense mechanism against invading viruses.
Inside prokaryote, restriction enzymes selectively cut up foreign DNA (=restriction digestion), meanwhile, host DNA is protected by modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage.
Together, these two processes form the restriction modification system.
Restriktionsenzyme:
Use?
More than 800 available commercially.
Routinely used for DNA modification in laboratories
a vital tool in molecular cloning
Shotgun Klonieriung?
method in cloning genomic DNA .
involves taking DNA to be cloned, cutting it either using a restriction enzyme or randomly using a physical method to smash the DNA into small pieces. These fragments are then taken together and cloned into a vector. The original DNA can be either genomic DNA (whole genome shotgun cloning) or a clone such as a YAC (yeast artificial chromosome ) that contains a large piece of genomic DNA needing to be split into fragments.
Shotgun Klonierung:
Schritte?
- a selected restriction enzyme cuts a specific base sequence everywhere it occurs in a chromosome or in cDNA
- the same enzyme cuts the same sequence in plasmid DNA
- the chromosomal DNA or cDNA fragments have sticky ends
- the plasmid DNA also has sticky ends
- the plasmid DNA and the foreign DNA are mixed in a solution with other enzymes that can seal them together
- the result: a collection of recombinant plasmids that incorporate foreign DNA fragments.
- host cells that can divide rapidly take up the recombinant plasmids
Shotgun Klonierung:
research more
Shotgun Klonierung:
research more
Vektoren?
Plasmide, um DNA Fragmente zu klonieren
Cloning Vectors
different types + size of insert accepted?
Plasmid: up to 15
Bacteriophage: up to 90
Bacterial artificial chromosome (BAC): 100 - 500
Yeast artificial chromosome (YAC): 250 - 2000
typische Klonierungsvektor?
pUC18
pUC18 Sequenzelemente?
pUC18 = Klonierungsvektor
Sequenzelemente:
- Polylinker = Multiple Cloning Site oder MCS (Bereich mit verschiedenen Restriktionsenzymserkennungssequenzen)
- Selektionsmarker (Ampicillin resistance gene)
- Marker fuer die Suche nach positiven Kolonien (LacZ gene)
- Bakterieller Origin of replication (oriR)
LacZ - wofuer in Klonierung?
fuer Blau/Weiss-Selektion (Visuelles Screening)
LacZ in Klonierungsverktor:
Funktionsweise?
LacZ = Gen fuer Beta-Galaktosidase - ein Enzym mit Laktose als Substrat.
X-Gal: ein artifizielles Glycosid und ein chromogenes Substrat für das β-Galactosidase. Galaktose + substituierte Indole.
Wachstumsmedium enthaelt X-Gal fuer visuelles Screening.
Blaue Kolonien:
= X-Gal –> Gal + X(blau)
= Beta-Galaktosidase ist da
= leerer Vektor, Klonierung hat nicht funktioniert
Weisse Kolonien:
= X-Gal bleibt so
= Beta-Galaktosidase nicht da
= enthaelt rekombinanter DNA
Nachteile des traditionellen Klonierens
1: verfügbare Restriktionsschnittstellen
limitierend
2: Multiple Arbeitschritte (e.g. Gelaufreinigungen
von DNA-Fragmenten)
3: Transfer von Inserts von einem Vektor zum
nächsten meist nicht trivial (e.g. auch um den
Leserahmen zu erhalten)
TA Klonierung?
- Restriktionsenzyme nicht noetig
- einfacher und schneller als traditioneller Klonierung
- Enden mit Thymine und Adenine, die unter Anwesenheit von Ligasen mit einander binden
- benutzt Taq Polymerase
Nachteil:
Weniger Hybride und weniger Effizienz
Topoisomerasen
Topoisomerasen vom Typ IA schneiden einen der beiden Einzelstraenge und fuehren den anderen durch die Luecke (entfernt Windungen)
Die sind gleichzeitig Endonuklease und Ligase
Topo Klonierung
zB Topo TA PCR Klonierung:
TOPO TA cloning Vector, mit Topoisomerase I recognition sites.
+
Taq-amplfied PCR Produkt mit A Overhangs
5 min at room temp –>
Ligation complete
Topoisomerase I is released
(noch video angucken zum verstehen)
Vorteile von TOPO Cloning?
• Per PCR kann man Insert exakt definieren –
keine mühsame Restriktionsschnittstellen-Suche
• Ligation hocheffizient (ca. 90%).
Gateway(TM) Klonierung:
Vorteile?
- Kompatibilität zwischen Vektoren
- Erhalt des Leserahmens
- *** Kein Bedarf von Restriktionsenzymen,
Gelaufreinigung, Ligation - Extrem schnell und ca 90% efficient - Hochdurchsatzmöglichkeit
also:
- Ease - att recombination sites used in combination with enzyme clonase mixes
- totally universal - all organisms
Gateway(TM) Klonierung:
basiert auf?
The Gateway system exploits the principle of site-specific recombination allowing the integration and excision of the λ phage back and forth out of a bacterial chromosome by itself.
Phage Lambda:
sehr ortsspezifische Rekombination (= Integration und Excision Reaktionen) von Phage in E. coli Genom.
Rekombination Orte (“att” sites) sind viel laenger als Restriktion-Sites –> sehr unwahrscheinlich dass sie in DNA zufaellig vorkommen
–> man kann mehrere DNA Fragmente von verschiedene Groessen parallel klonieren, mit den gleichen Enzyme.
Essentielle Merkmale des Lambda Systems:
Integrationsstellen? Enzyme?
Insertion und Exzision benutzt dieselben Sequenzen,
aber unterschiedliche Enzyme:
• Integration (attB x attP): Integrase (Int) und host integration factor (IHF) –> Es entstehen die Stellen attL and attR, die den
integrierten Prophagen flankieren
(Keine DNA Sequenzen gehen verloren)
Essentielle Merkmale des Lambda Systems:
Excisionssstellen? Enzyme?
Insertion und Exzision benutzt dieselben Sequenzen,
aber unterschiedliche Enzyme:
• Exzision (attL x attR): Integrase (Int); host integration factor
(IHF); Excisionase [Xis]: es entstehen attB in E. coli und attP in lambda
Gateway(R) Entry Clone
Research!
attB1-gene-attB2
+
Donor Vector (attP1-ccdB-attP2, KanR (Kanamycin resistance))
–> (mit BP Clonase(TM) II)
attR1-ccdB-attR2
+
Entry Clone (att1-gene-attL2, KanR)
Entry Clone: 90-99% correct clones on Kan (Kanamycin) Plates
Gateway(R) Expression Clone
Research!
Entry Clone(attL1-gene-attL2, KanR) \+ Destination Vector (attR1-ccdB-attR2, AmpR)
–>(mit LR Clonase(TM) II)
(Donor Vector (attP1-ccdB-attP2, KanR)
+
Expression Clone (attB1-gene-attB2, AmpR)
Expression Clone: 90-99% correct clones on Amp plates
Golden Gate Cloning
(seamless cloning)
Type II restriction enzyme
EcoRI -> digestion/religation
redigestable
Type IIS restriction enzyme
BsaI
digestion/religation
undigestable
Research more!
Golden Gate Cloning
Golden Gate Assembly Workflow
Research more!
Golden Gate Cloning
research more!
Golden Gate Cloning
research more
Golden Gate Cloning
research more
MoClo - Modular Cloning
research more
MoClo - Modular Cloning
research more
MoClo - Modular Cloning
research more
Gibson assembly
an alternative to restriction-based cloning
similar: Sequence and Ligase Independen Cloning (SLIC)
Gibson assembly
dsDNA fragments with overlapping ends
Add fragments to Gibson Assembly Master Mix
Incubate at 50 C for 15 - 60 Minutes
–> fully Assembled DNA
Gibson assembly
Gibson Assembly:
Exonuclease chews back 5’ ends
DNA fragments anneal
DNA Polymerase extends 3’ ends
DNA ligase seals nicks
Gibson assembly:
Assembly of multiple fragments using Gibson Cloning
Linear vector (REase-digested) + A, B (DNA inserts with 15 - 20 bp overlapping ends, PCR-amplified)
–>
Incubate at 50 C for 15-60 minutes
–>
Assembled DNA Plasmid
Transformation and Plating
Gibson assembly
Single-tube reaction with Gibson Assembly Master Mix:
- 5’ exonuclease
- DNA Polymerase
- DNA Ligase
Gibson assembly - Vorteile
- funktioniert auch bei sehr langen Fragmenten (1-2 kbp)
-
Gibson assembly
research more
The four fundamental invariable steps for cloning of any DNA fragment by classical restriction and
ligation-based approach
- DNA fragmentation with restriction endonucleases
- ligation of DNA fragments to a vector
- transformation
- screening/selection
Gateway Cloning (from Lit):
Gateway Cloning (from Lit):
welche Enzyme-Mix noetig?
- BP Clonase Mix und LR Clonase Mix
- bringt die Sequenzen hin und her zwischen Plasmide mit flankierende passende Recombination Attachment (att) Sites
Gateway Cloning (from Lit):
Multisite Gateway Cloning
The Multisite Gateway system includes vectors allowing to construct transcriptional units from two or three DNA fragments simultaneously.
This system was used for expression of several genes under the influence of a strong inducible or tissue specific promoter, for studies involving cis-regulatory sequence analysis, translation fusion, subcellular localization, for expressing tagged proteins.
Gene silencing, genomic fragment recombination, gene stacking and protein-protein interactions can also be methodically implicated using these recombinational Gateway expression cassettes.
Gateway Cloning (from Lit):
BP Clonase enzyme mix?
The BP clonase enzyme mix (“Invitrogen”/“Life Technologies”), consisting of the phage integrase and the integration host factor, transfers a DNA fragment of interest flanked by two attB sites into a donor vector (pDONR) carrying two attP sites.
Recombination takes place between the matching attB and attP sites and the DNA fragment is inserted into the donor backbone, resulting in an entry clone which is flanked by two L sites.
Gateway Cloning (from Lit):
LR Clonase enzyme mix?
The LR clonase enzyme mix (“Invitrogen”/”Life Technologies”), consisting of the integrase, integration host factor and the phage excisionase, catalyse the LR reaction, where the key substrates are entry clones.
The DNA fragment of interest flanked by two attL sites is transferred into a destination vector carrying two R sites.
Recombination again takes place between the matching attL and attR sites resulting in a novel expression clone flanked by attB sites.
Gateway Cloning (from Lit):
Assembly of entry clones can also be done by….
Assembly of entry clones can also be done by restriction and ligation of DNA fragments in vectors, where attL sites flank multiple cloning sites.
However, there is no such need, because the inventors of Gateway system engineered variants of the original attB, attP, attL and attR sites so that they will react specifically facilitating directional cloning.
Plasmid-Replikationskontrollarten
stringent vs relaxiert
recherchieren
