Genomic libraries Flashcards
What are the 2 main types of restriction nucleases for DNA manipulation?
- restriction endonucleases: making cuts at internal phosphodiester bonds at a specific sequence
- exonucleases: removing nucleotides from the ends of DNA/RNA molecules
What are the 3 main enzymes for DNA manipulation and their functions?
- DNA polymerase: synthesise new polynucleotides complimentary to an existing DNA/RNA template
- nucleases: degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next
- ligases: join DNA molecules together by synthesising phosphodiester bonds between nucleotides
How can you prevent a vector self-ligating? (3)
Use 2 non-compatible restriction enzymes
And 2 different restriction sites
Modify the DNA ends
Name 3 end-modification enzymes and their functions
- alkaline phosphatase: removes phosphate groups from 5’ ends of DNA, preventing them from ligating to each other
- t4 polynucleotide kinase: adds phosphate groups to 5’ ends - end labeling
- terminal deoxynucleotidyl transferase: template independent DNA polymerase. Homopolymer tailing.
Name 3 common engineering strategies to maximise the production of a recombinant molecule
1) use of 2 restriction enzymes
2) alkaline phosphatase treatment
3) fragment modification
When adding sticky ends to blunt ends:
4) DNA linkers
5) making DNA adapters using polynucleotide kinase
6) homopolymer tailing using terminal deoxynucleotide transferase
Name the 6 common vectors in order of their cloning capacity.
Plasmids, phagemids, lambda phages, cosmids, bacterial artificial chromosome, yeast artificial chromosome.
What is complete or partial DNA digestion and why is partial digestion favoured?
Complete - the restriction enzyme cuts the DNA at every target site, giving differing sized fragments
Partial - the restriction enzyme cuts fewer sites producing larger, overlapping fragments.
Complete may disrupt gene expression, whereas partial has a higher chance of gene being expressed. Overlapping fragments allows the genes to be mapped for libraries etc.
What is chromosome walking?
A sequencing method for DNA that is too long to be sequenced in a single sequence. To find, isolate and clone a particular allele in a gene library.
It starts with an already identified gene, to make a probe, which can be used to walk towards another gene that you want to clone but don’t know the sequence of on overlapping DNA.
What are c DNA libraries?
A collection of clones that represent all expressed genes in a specific tissue
What are expressed sequence tags? How are they useful?
Partial sequences of clones, used to identify genes based on sequence homologous or primer design. Cheap approach
What are the 3 ways of ligating cDNA into cloning vectors?
1) direct blunt end ligation (very inefficient)
2) addition of adaptor or linker
3) homopolymer tailing
What is homopolymer tailing?
Ligating method
Adds a string of nucleotides to 3’ end (requires vector to have a complementary tail added)
- use klenow polymerase to fill gaps
What is T/A cloning and what is it useful for?
Rapid cloning of Pcr products
Useful if you are amplifying a specific gene from your cDNA sample
What are the main problems with c DNA libraries?
- library sequences will not contain promoter regions
- if gene is expressed at low levels, very little mRNA will be present, making it difficult to find in library
- mRNA expression may not infer functional protein expression
