31. Cloning Flashcards
1
Q
what are restriction enzymes?
A
- growth in some bacteria restricted
– phage DNA is destroyed - bacteria have endonucleases that cut double stranded DNA at specific sequences
- bacterial DNA not affected
– bacteria have enzymes which add methyl groups to specific sequences in bacterium’s DNA making immune to restriction
2
Q
what is the importance of REs?
A
- bacteria have epigenetic inheritence of DNA methylation patterns
– methylation patterns of their DNA is transferred to dividing cells
3
Q
how do viruses escape restriction?
A
- accidental methylation of DNA, thereafter methylated every time it is replicated
– changes / mutations in viral DNA sequences
4
Q
what are RE recognition sites?
A
- often palindromes
- lengths of recognition sites (4-8 bp length)
– determine how often may occur in viral genome - creation of recombinant pieces of DNA
– consider RE which doesn’t restrict (cut) within gene sequence to be cloned - isoschizomer
– RE with same recognition sequence
– different name because they were isolated from different bacteria
5
Q
what are sticky and blunt ends on REs?
A
- name of RE gives species of bacterium isolated from
- EcoRI
– E.Coli RY13 strain
– roman numeral (I) idnicates first RE identified from the organism
6
Q
what are the different types of cloning?
A
- two sticky ends
- two different but compatible ends
- one sticky end and one blunt end
- both blunt ends
7
Q
what are the requirements for cloning with two sticky ends?
A
- compatible sticky ends
- directional cloning
- efficient insert-vector ligation
- low vector self-ligation
- intact recognition sites of ligated restricted enzymes
8
Q
what are the requirements for cloning with two different but compatible ends?
A
- compatible sticky ends
- directional cloning
- efficient insert-vector ligation
- low vector self-ligation
- recognition sites of original restriction enzymes may be destroyed after ligation
9
Q
what are the requirements for cloning with one sticky and one blunt end?
A
- directional cloning is maintained
- ligation of blunt end may be less efficient
10
Q
what are the requirements for cloning with two blunt ends?
A
- ends are compatible
- end sequences are modified
- directional cloning is lost
- ligation may be less efficient
- vector self-ligation is high
11
Q
what are the considerations needed when choosing RE sites?
A
- RE must not be too close together on vector or in the multiple cloning site
– not all can cut if too close - if can’t find suitable site
– use PCR add sites
12
Q
what is DNA ligase?
A
- joins two nucleic acid fragments by an enzyme
– ligation in lab, performed by T4 DNA ligase - ends of DNA fragments joined together by formation of phosphodiester bonds between 3’-hydroxyl of one DNA strand anf 5’-phosphoryl of another
– co-factor required, usually ATP / NAD+ as reducing agent
13
Q
how are primers designed to include restriction sites?
A
- use PCR to add additonal sequence information to target sequences
– introduce sequences recognised by REs using PCR - non-complementary sequences always added to 5’ end of primer
– generates compatible ends to our DNA, allowing insertion into vectors - design primers ligate a DNA fragment into plasmid vector using EcoRI and HindIII restrictions sites
– to create recombinant plasmid (plasmid containing foreign DNA fragment)
14
Q
what is TA cloning?
A
- PCR performed with Taq polymerase
– enzyme adds additional A base to 3’ end of amplified product
– result of lack of proofreading capability of this enzyme - as A will complementary base pair with T
– commercial vectors with compatible A overhands exist
– can be used for cloning of Taq generated PCR products
15
Q
what is blunt cloning?
A
- polymerase with high proofreading capability do not add A to 3’ end of PCR product
- PCR performed with Pfu results in blunt ended PCR products
- commercial blunt cloning vectors allow for direct cloning of Pfu generated PCR products
- no directionality
