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Cloning Approaches
Sequence-Based Cloning Appraches
| Question | Answer |
|---|---|
| What is VECTOR? | OOH YEAH BABY MAGNITUDE AND DIRECTION VECTORRRRR |
| What is a VECTOR? | DNA molecule into which foreign DNA is inserted for propagation in host cell |
| Essential features for ALL vectors? (3) | 1. Can incorporate 'foreign' DNA 2. Can be readily introduced into host cell & selected 3. Can replicate in host cell |
| How is a bacterial host prepared for a DNA cloning experiment? | 1. Disablement of host (for safety) 2. Stable maintenance of transformed DNA 3. Efficient 'transformation' by plasmid DNA |
| How is the bacterial host made safe for DNA cloning? | Auxotroph used - only grows on growth medium supplied in lab |
| How is transformed DNA stably maintained? (Bacterial host DNA cloning) | Use of mutants in recombination genes to avoid rearrangements |
| Example of a mutant recombination gene used to maintain stable transformed DNA? | recA |
| What is cloning? | Joining a given piece of DNA to functions that enable replication of that piece of DNA |
| How is the fragment of interest linked to the vector DNA molecule? (cloning) | Phosphodiester bonds |
| What does vector choice depend on? | Size, availability, screening |
| Common vectors with E.coli (2) | 1. Plasmid vectors 2. Bacteriophage lambda vectors |
| When can the vector molecule replicate? | When it's introduced to the host cell |
| Difference between plasmids and lambda vectors? | Plasmids replicate along with host cells, lambda vectors replicate as lytic viruses - killing host & packaging into virions |
| Vector DNA specific key steps in DNA cloning experiment? (5) | 1. Purify vector plasmid DNA 2. Restriction digest vector DNA with suitable RE(s) 3. Alkaline phosphatase treat 4. Heat inactivate enzymes, 65°C 10 min 5. Agarose gel purification of vector fragment (OPTIONAL) |
| Why would you skip the alkaline phosphatase treat? | If the vector was digested with 2 different REs |
| Insert DNA specific key steps in DNA cloning Experiment? (4) | 1. Purify insert DNA from a suitable source 2. Digest insert DNA with REs with compatible ends to digested vector 3. Heat inactivate enzymes, 65°C, 10 min 4. Agarose gel purification of insert fragment |
| Suitable sources for insert DNA? (3) | 1. Genomic DNA 2. cDNA 3. Recombinant vector |
| Steps in DNA cloning after the ones specific for insert/vector? (FIRST 3 STEPS!!) | 1. Ligate vector and insert DNAs in ligase buffer, ligase and ATP 2. Transform into competent E.coli 3. Grow overnight on agar containing appropriate antibiotic to select for clones containing vector |
| Steps in DNA cloning after the ones specific for insert/vector? (LAST 2 STEPS!!) | 4. Pick a few colonies for colony PCR/purify plasmid DNA & perform RE digests to identify putative recombinants 5. DNA sequence insert of putative recombinants |
| What is in the traditional toolkit for DNA cloning? (4) | 1. Restriction enzymes 2. DNA ligase 3. DNA polymerases 4. Alkaline phosphatases |
| What do restriction enzymes (REs) do? | cut DNA at specific sequences - often leave 5'/3' overhangs/sticky ends |
| DNA ligase function? | joins DNA strands via 5'P and 3' OH, following base pairing, to form recombinant DNA |
| DNA polymerase function? | Produces blunt ends by filling in 5' overhangs/removing 3' overhangs |
| Alkaline phosphatases function? | Removes 5' monophosphate groups from DNA preventing ligation of that strand |
| restriction enzyme naming??? | 1st letter = genus 2nd & 3rd = species Roman numeral = which enzymes from that organism |
| What is PvuI | Proteus vulgaris I |
| Name 2 bacterial REs | EcoRI & EcoRV E.coli R strain 1st/5th enzyme |
| How many types of REs are there? | 5 |
| Type II REs? (3) | 1. Most cleave within recognition site 2. Some cleave outside but CLOSE to recognition site 3. Restrict infection of bacteria & archaea by phage |
| BamHI? | Type II RE that cleaves within recognition site |
| BsaI? | Type II RE that cleaves outside but close to recognition site - used in Golden Gate cloning |
| Length of a recognition site in cloning? | 4-8 base pairs |
| What does length of recognition site determine? | How often site will appear by chance in any given genome |
| What is a neoschizomer? | RE that recognises the same sequence |
| What is an isoschizomer? | Re that recognises and cleaves in the same position |
| What does the ligase buffer contain? | ATP and DNA ligase |
| Where does most purified DNA ligase used in DNA cloning originate from? | Bacteriophage T4 |
| What can a Klenow fragment be used for? (2) | 1. Fill in 5' overhangs in presence of nucleotides 2. Trim 3' overhangs to form blunt ends |
| What is a Klenow fragment? | Molecule that can produce recombinant DNA molecules - Proteolytic product that retains the polymerization fidelity of the native enzyme without degrading 5' termini |
| Why were Klenow fragments replaced by Taq polymerase? | They are not thermostable |
| What are plasmids? | Circular dsDNA molecules - separate from a cell's chromosomal DNA |
| How have E.coli plasmids been engineered to optimise use as vectors in DNA cloning? (4) | 1. Length reduced 2. Replication origin 3. Selectable marker 4. Cloning site(s) |
| What is pUC19 and what does it have(2)? | typical plasmid cloning vector 1. Multiple cloning site (MCS) 2. Short section of lacZ gene |
| What does plasmid AmpR do? | Inactivates ampicillin |
| What are competent cells? | Cells which CAN take up DNA from the environment |
| What are transformants? | Cells that DO take up DNA from the environment |
| Method of identifying plasmids? | Blue-white selection |
| What does blue-white selection detect? | b-galactosidase production |
| What does b-galactosidase do? | Cleaves lactose into glucose & galactose |
| Blue white selection, what colour are clones without the insert? | Blue |
| What must be produced by the cell for blue-white selection to work? | Alpha fragment & b-galactosidase |
| What is the pET system? | The most powerful system to produce recombinant protein in E.coli |
| What is the purpose of the His tag in a pET vector? | To facilitate easy purification of protein by immobilised metal affinity chromatography |
| How many bases does the fwd primer need at its 3' end (of coding sequencing) | 18-30 bases (from start codon of gene of interest) |
| Protease Specific cleavage?? | IRREVERSIBLE |
| What does Golden Gate Assembly allow? | Ordered assembly of a vector & DNA fragments into single construct suitable for direct transformation of bacterial host |
| What type of enzymes does Golden Gate use? | Type II restriction enzymes (eg BsaI) that cut outside recognition sites |
| Golden Gate Assembly method please? (4) | 1. 2 fragments have BsaI sites but in opposite directions 2. BsaI cleavage generates complementary 5' protruding ends 3. 5' ends ligated together with DNA ligase 4. Final assembly no longer has type II RE sites - no further cutting of BsaI is possible |
| How is Gibson Assembly different from Golden Gate? | Gibson does not involve REs |
| Gibson Assembly benefits please? (5) | 1. Multiple fragments can be assembled in one reaction 2. Can use long fragments (>3kb) 3. Scarless 4. Any insert fragment w/ apt overhangs can be ligated into any vector 5. Not limited by factors of trad. cloning |
| Gibson Assembly method please? (4) | 1. 5'-exonuclease activity produces ss segments (3' overhangs) - 'chews back' 5' ends 2. 3' overhangs are complementary to each other - pairs of template and primer produced 3. DNAP fills in gaps 4. DNA ligase links DNA fragments |
| Limits of Traditional cloning please? (4) | 1. Multiple reactions 2. Restricted to joining 2 fragments in single ligation 3. Limited by suitable restriction enzymes 4. Difficult to join larger fragments |
| Why is it difficult to join larger fragments in traditional cloning? | Lower concentration of ends, less self ligation |
Created by:
rubyroo
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