TD: DNA Restriction & Ligation Flashcards
Describe the steps involved in DNA cloning
Cloning of a gene occurs in the following stages:
- The gene (target DNA) requires to be isolated and rejoined with cloning vector DNA (such as a plasmid).
- The joined DNA, termed recombinant DNA, must be placed into an appropriate host such as E. coli to allow expression.
- Clones expressing the gene must be recovered.
What is the host recognition and modified system response?
- Bacteria have a crude defensive/immune system that operates to protect the cell eg against viral attack.
- This is the host restriction and modification system which functions specifically to cleave (or break) non-host DNA.
- The enzymes catalysing non-host DNA cleavage are termed restriction endonucleases (RE).
- Host DNA is protected from cleavage by addition of methyl groups, catalysed by modification methylase.
Restriction endonuclease recognition sites - describe these
Describe restriction endonucleases and the process of forming fragments suitable for cloning
- Each bacterium harbours at least one RE.
- Named after the host from which it is derived eg EcoR1 from E.coli strain R.
- Three major categories of RE exist differing in mode of action, classified as types I, II, or III.
- Only type II RE are suitable for cloning.
- Recognise unmethylated target sequences in DNA and break both strands of the molecule within this sequence ie they cleave DNA precisely.
- This activity can produce discrete DNA fragments of defined length and sequence.
- Such fragments are suitable for cloning.
Each type of RE has a different target site - what are the common characterisitcs of this sequence?
What happens during the cleavage process?
- Each type II RE has a different target site, commonly a precise hexanucleotide sequence which is symmetrical in nature.
- Upon recognising the target site, the RE binds and catalyses cleavage of specific phosphodiester bonds.
- As both strands are cleaved the fragments separate as the intrachain H-bonds are too weak to hold the two fragments together.
What is the restriction sequence for EcoR1
•The RE EcoR1 recognises the sequence:
5’…….G A A T T C…….3’
3’…….C T T A A G…….5’
•EcoR1 makes single stranded breaks four nucleotide pairs apart, in opposite strands of the target sequence, releasing fragments with protruding 5’ ends:
5’…….G 5’ A A T T C…….3’
3’…….C T T A A 5’ G…….5’
What are the protruding ends commonly termed with EcoR1 fragment formation?
What are other ends termed as?
- These protruding ends are commonly termed sticky ends as the fragments can associate transiently by H-bonding.
- Importantly, DNA fragments from different sources can be joined via complementary sticky ends.
- Other type II enzymes can function either to produce 3’ protruding ends eg Pst1,or blunt ends eg Sma1.
Describe the joining of DNA fragements
- The process of joining DNA fragments is termed ligation, catalysed by the enzyme DNA ligase.
- The H-bonds briefly hold the complementary sticky ends together allowing phosphodiester bond formation.
- Cloning of blunt ended fragments is more difficult as they cannot associate by H-bonding.
- Homopolymer tailing is one method developed to improve cloning of these blunt ended fragments.
Describe using homopolymers to help the ligation process of blunt ended fragments
- The enzyme terminal transferase catalyses addition of deoxynucleotides onto the 3’end of linear DNA fragments.
- Addition of complementary homopolymer sequences onto the end of target DNA and vector DNA allows these to associate via H-bonds.
How is the recombinant DNA after ligation inserted into a host cell for cloning?
Describe this process
- Following ligation the cloning vector is transformed into the host cells e.g. E. coli, yeast or mammalian cells (transfection).
- Transformation of E. coli host cells with the cloned DNA can be achieved by heat shock or electroporation.
Cells are allowed to recover then the success of transformation is assessed
What are molecular cloning vehicles?
Most common used?
- •A cloning vector is:
- -a DNA molecule capable of harbouring foreign DNA,
- -able to replicate within the host independent from the genome.
- •Several vector types are available, but plasmids are most commonly used with bacterial hosts.
What are plasmids?
Properties associated with ideal plasmid cloning vectors are?
- Plasmids are small, circular, naturally occurring DNA molecules.
- Properties associated with ideal plasmid cloning vectors are:
- Low molecular weight - smaller plasmids permit larger amount of DNA to be cloned and allow easier host transformation.
- Single restriction sites - plasmid must be cut open to insert DNA being cloned.
- If more than one restriction target site exists more that one fragment is produced which causes problems.
- Selectable genotype/phenotype - there is a requirement to distinguish transformed from non transformed cells ie cells with recombinant plasmid from those without.
What is the problem with plasma vectors?
•The main problem with plasmid vectors is a limitation on the amount of DNA that can be cloned ie 10.0 kb vector and cloned DNA max.
The plasmids contain specific markers such as AB resistance which help us identify which cells have been transformed.
See back of card
Describe selection of recombinat clones
- During insertion of recombinant DNA only ~1 in 10,000 cells in the bacterial population are successfully transformed.
- The cells expressing the cloned gene must be isolated from the population.
- Methods used to select recombinant clones include genetic, nucleic acid hybridisation (NAH) or immunological approaches.
Describe the growth of ecoli in a medium with both glucose and lactose. What does the graph look like?
- When E.coli grows in medium containing both glucose and lactose a biphasic logarithmic growth curve is observed.
- Glucose is exclusively catabolised until exhausted.
- Only then is lactose used.
- Only when E.coli needs to metabolise lactose are the necessary enzymes synthesised.
- Hence, E.coli does not waste resources synthesising enzymes unnecessarily.
- The enzymes are b-galactosidase and lactose permease, produced by the lacZ and lacY genes, respectively.
- Lactose permease functions to transport lactose from the medium into the cytoplasm.
- b-galactosidase catalyses cleavage of lactose into glucose and galactose.
- Immediately preceding exhaustion of glucose only a few molecules of b-gal and lac permease are present.
- After glucose is consumed, the quantity of these enzymes increase over 1000-fold.
- Lac repressor protein is continuously manufactured, and in the absence of lactose binds the operator region of the lac operon.
- Lac repressor binding prevents transcription by blocking RNA polymerase access to the promoter site ie negative regulation.
- Once glucose is exhausted, E.coli will switch to catabolism of lactose through inhibition of lac repressor binding.
- Initially some lactose is converted to allolactose, the lac operon physiological inducer.
- Lac repressor is an allosteric protein, with inducer binding triggering a structural change resulting in dissociation from the lac operator.
- RNA polymerase can then initiate transcription.
Describe Catabolite Repression
- Glucose controls expression of the lac operon through catabolite repression.
- When glucose is abundant cytoplasmic cAMP levels are low, and once glucose is depleted cAMP levels rise.
- cAMP is bound by cAMP receptor protein (CRP), this complex then attaching to the lac operon promoter region.
- This allows transcription, but only in the
- Binding of CRP-cAMP complex is necessary to turn on lac operon transcription.
- CRP-cAMP bends the lac promoter into a shape that RNA polymerase efficiently binds to.
- In this way CRP-cAMP forms part of the binding site for RNA polymerase.
absence of lac repressor bound to the operator ie positive regulation.
