enzymes used in molecular cloning Flashcards
what’s molecular cloning
-A set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules
what’s recombinant DNA
-A type of DNA sequence that is composed of sequences from two or more different sources or organisms, such as synthetic (lab-made) sequences and microorganisms
Why do we want to carry out molecular cloning
-We use molecular cloning to isolate a specific region of DNA that we are interested in
=An entire gene
=The coding sequence (CDS) of a gene
=A promoter region
-Downstream we may want to:
=Sequence a gene
=Analyse mutant vs WT genes (cancer or not)
=Make mutations in the DNA manipulating DNA
=express and purify the protein
Steps of molecular cloning:
1)First step is to make recombinant DNA.
2)In molecular cloning, one of the pieces of DNA is a vector (discuss what this is in a moment – basically a carrier).
3)Cut and paste vector to the DNA fragment of interest.
4)Recombinant DNA is not much use unless we can isolate a single species and make more of it. Put it into host – E. coli (“transformation”). Acts like a factory.
5) Selection and host replication – lots of progeny, all containing recombinant DNA.
what’s a vector
-Basically a carrier for your favourite gene
-Insert DNA can probably get into a host cell – but it won’t last long there
-Vector has features that enable it to get into a host and maintain / replicate itself there
-Anything DNA that is part of the vector will be maintained / replicated too (like your insert, for example)
-capable of: Getting into the host, Replicating in the host and Being maintained over generations
what are 3 most common features in vectors
-Origin of replication
-Selectable marker
-Multiple Cloning Site
-commonly used in plasmids
vectors: selectable marker
-Survival of host cells that are carrying your plasmid
vectors: multiple cloning sites
-Where to clone your gene
-Restriction enzyme sites- clone site of interest, cuts open in various different ways
vectors: origin of replication
-Independent replication inside the host
-allows vectors to be inserted to be replicated
uses and limitations of plasmids as vectors
-Size of insert (limited in plasmids)
-Copy number (up to 500)
-Application
How do we clone our DNA into the vector and what is needed for this
-Something to cut DNA in a specific place
-Something to stick DNA back together again
-A way to prepare your insert: Cut, Amplify, Purify
-Modify the DNA ends
-Way to stick them back together again
restriction and modification
-Observed that phage grown in one bacterial (E. coli) host often failed to grow in another – growth was restricted.
-However, some rare progeny were able to grow in the new host due to some sort of modification which allowed them to do so.
-This modification was observed to be reversible – not a permanent change
-Hypothesis – nuclease + some sort of DNA modification
Proved when first enzymes characterised
-different bacteria have different restriction enzymes
why is DNA not degraded when E-Coli is infected with different strains in restriction and modification
-methylation of host DNA at those sequences.
-Sometimes a rare piece of invading phage DNA acquires the correct methylation pattern – now protected from degradation
-Phage can produce progeny
how many restriction enzymes are there
-thousands
what do restriction enzymes do
-Recognise a short, specific DNA sequence
-Broadly classified into three/four types
=Types I and III cleave DNA at random far away from recognition sequence.
=Type IV cleave modified DNA.
=Type II are the useful ones, They cut DNA at a defined position (very specific), either within or near to their recognition site
how do we name restriction enzymes
-First 3 letters = species (E. coli)
-4th letter = strain (RY13)
-Roman numeral = which enzyme from that species/strain (at least 5!)
type II restriction enzymes
-Most widely used ones are protein homodimers.
-DNA sequence is usually palindromic.
-Recognise specific DNA sequence (usually 4-8bp).
-Can generate overhangs (5’ or 3’) or blunt ends.
-Cleavage generates 5’ phosphate and 3’ OH groups
-Most useful Type II REs are often homodimers – two identical polypeptides: =Sequence-specific – single nucleotide change eliminates activity. (All REs recognise a specific sequence. (6bp cutters most commonly used in molecular biology).
=Palindromic – reads same 5’-3’ on each strand.
=Ends can be blunt (SmaI) or overhanging (HindIII) – also called “sticky” ends
how do restriction enzymes work
-This is BamHI
-Initial binding is non-specific: looser, catalytic site not involved (no specific cutting)
-Enzyme then moves along DNA: it can “slide” for short distances but can also jump or hop over longer distances if it doesn’t encounter a specific site
-Recognition of a specific site-> conformational changes (enzyme and DNA). Exact mechanism not yet known
Ends have 5’-phosphate and 3’-OH groups
what does the overhanging ends being sticky mean
- can base pair with matching overhanging ends
-Any overhanging end generated by EcoRI is compatible with any other.
-Can cut vector and insert with same enzyme->compatible overhanging ends
why is pairing not permanent when compatible sticky ends stick back together
-Complementary sticky ends can come together and hydrogen bonds form between the complementary base pairs. Temporary interaction
-The sugar-phosphate backbone is not continuous and something has to seal this gap – form a new phosphodiester bond
-This is why the phosphate and OH are important
-= DNA ligase
what’s the The ligation reaction
-First the ends have to interact with each other (hydrogen bonding)
-Inefficient – lower temperatures help (slower molecular movement; stabilises H-bonds for sticky ends)
-If step 1 happens for long enough, DNA Ligase catalyses the formation of a phosphodiester bond (between phosphate and OH groups)
-Better at higher temperatures (25 C optimal)! –Compromise – 1h at 16 C / overnight at 4 C
-ligation can also happen between blunt-ended fragments, Don’t have to be sticky ends. Ligation of blunt-ended fragments is much less efficient (no temporary hydrogen-bonding between complementary ends).
whats the DNA ligase mechanism
-AMP is transferred to a lysine residue in the enzyme’s active site (from ATP – the cofactor).
-AMP is then transferred to the 5′-phosphate.
-The AMP-phosphate bond is attacked by the 3′-OH, forming the covalent bond and releasing AMP.
-ATP is required to replace the AMP used in the reaction (i.e. is a cofactor)
potential issues with PCR
-There might not be convenient restriction sites.
-one might not have enough DNA.
-My DNA might be mixed in with lots of other DNA molecules
-The vector has complementary ends – it might ligate to itself (very likely in fact – closer together)
-Modify vector ends – phosphatase treatment removes 5’ phosphate – no phosphodiester bond can be formed
-Gene may insert in wrong orientation
-Use more than one enzyme (also solves first problem) for each end
Addition/removal of the 5’-phosphate in modifying DNA ends
-5’ phosphates are required for ligation. No phosphate = no ligation
-If there are only 5’ phosphates on one strand at each end then only one of the two DNA strands is going to form the phosphodiester bond.
-The other strand we will have a ‘nick’ in the DNA but this will be repaired by host enzymes once inside a bacterial cell
adding a 5’ phosphate in modifying DNA ends
-If there is no phosphate, we need to add one for ligation to work
-PCR products usually don’t have 5’ phosphate groups – ends need to be phosphorylated for ligation to be successful
-Restriction enzyme cut DNA has a phosphate
-Polynucleotide kinase will catalyse phosphorylation of 5’ ends (from ATP)
Removing the 5’-phosphate in modifying DNA ends
-“Sticky” ends can still base pair via hydrogen bonding but ligation can no longer occur
-Can be used to prevent self-ligation of your vector
-Removing a phosphate can prevent unwanted ligation (e.g. self ligation of a vector during cloning).
-Treat cut vector with CIP => removes 5’ phosphate. Sticky ends can still associate but no covalent bond is formed. Ligation cannot occur
removing a DNA overhand in modifying DNA ends
-blunt end cloning might be necessary
-destroy restriction enzyme sites
-less commonly used but can be useful
what’s used for the DNA ends modification
-T4 DNA polymerase or DNA polymerase I, large fragment - for filling the 5’ overhang (polymerase) and removing the 3’ overhang (exonuclease activity)
-for removing the 5’ overhang its mung bean nuclease
what’s happens in transformation
-Electroporation
=Brief pulse of high-voltage
-Chemical Transformation
=Chemically treated E. coli
=Subject to heat-shock
=Causes cell membrane changes that allow uptake of DNA
-Potential problem – this is not 100% efficient
if the bacteria have no plasmid then how do you select the ones that do
-Use the selectable marker on your vector.
-Typically, it is an antibiotic resistance gene that allows bacteria to grow on a place containing that antibiotic.
-Each single bacterium will form a colony of identical bacteria containing the plasmid
-Successfully transformed bacteria will contain the vector. As the cells divide, so does the plasmid – hence the name “cloning”
what can empty plasmids do
-transform host cells – selectable marker cannot do this for you.
-Your cloning strategy should minimise this
-Still need to screen transformants – restriction enzymes / PCR
