Lecture 2 Flashcards
Restriction Endonucleases (REs)
cleave double stranded DNA by breaking phosphodiester bonds
What is the role of restriction endonucleases in nature?
protect cell from infection by foreign DNA
ex: bacteriophage (virus)
What is the type of Restriction enzymes we use and describe?
Type II:
- smaller usually homodimers
- only require Mg++ for activity
- unlike type 1, cleave DNA at specific sites
- *are used for cloning
Restriction sites
- recognition sequence on DNA that restriction enzyme binds to and cleaves the phosphodiester bond
- usually palindromic (read the same 5’ to 3’ on both strands), but the sites can vary both in nucleotide sequence and length
- Different enzymes recognized distinct sites
Restriction digestion (or digest)
in cloning, adding a specific restriction enzyme (or enzymes) to specific DNA to obtain predictable or inter predictable DNA fragments
Blunt ends
a cut at the center of dyad= blunt ends
NNNGG/ CCNNN
NNNCC/ GGNNN
Sticky (cohesive) ends
a cut left of center produces cohesive ends with 5’ overhangs or to right of center, 3’ overhangs
How do you determine the frequency of a restriction site?
frequency of cleavage sites in DNA: 1/4^n where n=length of restriction site
4bp site- frequent cutter
8bp site- very infrequent
Restriction enzyme nomenclature
first letter= first initial of genus from which the enzyme was isolated
second and third letters= first two initials of species (first three letters are italicized)
Fourth letter(optional)= is letter of strain, not italicized
Perfect isoschizomers
enzymes that recognize the SAME DNA SEQUENCE (and cut the same), but are isolated from different organism
Imperfect isoschizomers
enzymes that recognize the same DNA sequence, but one enzyme recognized UNMETHYLATED DNA and one restricts only methylate DNA.
Neoschizomers
enzymes that recognize the same sequence, but CUT DIFFERENT NUCLEOTIDES
When setting up a RE digestion reaction: how do you determine what buffer to use?
- buffers are supplied at 10X with the enzyme
- some enzymes also require the presence of BSA(now it comes already spikes in the buffer)
- if performing a double digest, look at each enzymes activity in the various buffers, or look at chart at neb.com
Enzyme activity (unit)
the amount of enzyme required to digest 1 microgram of DNA in a 50 uL reaction in 1 hr
When setting up a RE digestion reaction: how do you determine how much enzyme you should add to your reaction?
- usually add 5 to 10 fold excess than needed
- NEVER let volume of the total enzyme exceed 10% reaction volume (adding to much enzyme can lead to star activity-ex: cutting sequence other than recognition site)
- ALWAYS add last to your reaction
What is a expression vector and what are some of its basic features?
-have basic features in common with cloning vectors, but also carry components necessary to transcribe and translate your gene of interest
Basic features:
- origin of replication- often lower copy number than cloning vector
- selectable marker (ex: antibiotic resistance)
- Multiple cloning sites
usually no screenable marker, because these are rarely compatible with expression of the inserted gene
- promoter- drives transcription of gene
- Ribosome binding site-present near start of transcript and signals ribosome to bind and scan for start codon
- start codon- nucleotide triplet (codon) that signals start of translation
- multiple cloning site- désigned to allow insertion in a predictable reading frame and orientation
- fusion tag- can aid in purification, detection, etc of protein- not always desired/ present because it changes the protein.
What does a restriction endonuclease digestion do?
breaks a phosphodiester bond
DNA ligase
creates a phosphodiester bond
catalyzes covalent bonds formation between 3’OH and 5’ PO4 on DNA
Blunt ends characteristics
- any two blunt ends can be ligated
- less efficient, but just give it a little more time
Sticky ends characteristics
- very efficient in proper buffer conditions
- Must be compatible (have complimentary base pairs) in over to be ligated together
How are compatible cohesive ends produced?
produced by restriction enzymes that recognize different sites, but creates staggered ends that are complementary
Can hybrid sites that are formed by ligation be recleaved?
NO
During ligation with insert, plasmid vectors have a tendency to relegate upon themselves (to generate original circular plasmid) rather than with an insert. How can we minimize this?
pohophatase treatment
Forced cloning
Does ligation occur spontaneously?
No, ligation only occurs in the presence of an enzyme such as ligase, it does NOT OCCUR SPONTANEOUSLY
Phosphatase treatment
- useful if cloning with only one enzyme and in blunt-end cloning
- treat ONLY the vector with alkaline phosphatase (AP)
- AP removes 5’ phosphates from ends of DNA
- vector cannot relegate upon itself; it can only ligate to insert
- possible disadvantages: insert can go in either direction
- MUST heat inactivate AP before ligation!!
- after ligation, each strand will have a nick reparable in cells
Forced (directional) cloning
- Double digestion with two different enzymes that leave incompatible ends will prevent vector from re-ligating (if stuffer fragment is removed)
- Insert is cut with same two enzymes use to cut the vector
- Another advantage is that the orientation of the insert is forced
In our project what will our insert and vector DNA be digested with?
NcoI and NotI restriciton enzymes
How does DNA move on a gel?
DNA is negatively charged, so it moves toward the anode. The agarose gel matrix retards large molecules more than small, so small fragments move a further distance through the gel.
Gel Red
- will be used instead of ethidium bromide for the visualization of DNA under UV light
- Gel red binds specifically to DNA and is excited by UV light; therefore when the gel is exposed to UV light you can visualize the DNA
DNA loading buffer
also called loading dye or sample buffer
- is added to DNA sample before loading the gel
- contains glycerol which is heavier than TBE or TAE buffers
- contains ore or more dyes in it
