DNA extraction and enzymes Flashcards
What 3 things must the DNA be separated from during extraction?
- Lipids
- Proteins
- Metal cations
Why must metal cations be removed?
They are used as cofactors by DNAses.
Which reagents dissolve lipids? Give 3 examples.
- Phenol
- Chloroform
- Detergents
Which 4 reagents remove proteins by denaturation and to what degree?
- Phenol - strongly denaturing.
- Chloroform - weakly denaturing.
- Detergents - strongly denaturing.
- Guanidine thiocyanate - strongly denaturing.
Which 2 reagents are hydrophobic?
Phenol and Chloroform.
Which reagent is hydrophilic?
Detergent.
Which reagent digests protein as oppose to denature it?
Proteinases.
Guanidine thiocyanate is said to be a chaotropic agent - what does that mean?
It causes DNA to bind reversibly to silica, allowing it to be separated.
What does EDTA do?
Chelates metal cations so they cannot be used by DNAses as cofactors.
Are pH buffers needed in DNA extraction?
Yes.
What does alcohol in combination with various salts do to DNA?
Causes it to form a solid ppt.
What do commercial resins do?
Selectively bind to DNA/proteins.
What are nucleases?
Enzymes that cut DNA.
What is an exonuclease?
An enzyme that removes nucleotides from the end of a molecule.
What does ribonuclease A do?
Attacks ssRNA on the 3’ end of pyramidine nucleotides. This causes unhybridised RNA to be removed.
Under what 2 conditions must enzymes be stored and why?
- -20 degrees to prevent denaturation
2. In glycerol to prevent oxidation
What are restrictions endonucleases (REs)?
Enzymes that cleave DNA internally.
What 3 major conditions are required for REs to work?
- Mg2+
- pH 7-8
- 37 degrees
Which RE has a different optimum temperature and what is it?
Taq1 requires 65 degrees.
Where do REs cleave DNA?
At palindromic recognition sequences.
Does each RE have a specific recognition site?
Yes.
What are the 3 major classes of RE and how do they differ?
- Type I - cleave approx. 1000bp away from the recognition site.
- Type II - cleave at the recognition site.
- Type III - cleave approx. 24-26bp away from the recognition site.
Where do REs come from?
Isolated from bacteria that use then as defence against viral infection.
Why is bacterial DNA unaffected by REs?
Bacteria produce methylases with the same recognition sites as the REs. They then methylate the DNA to prevent it from being cut.
How are REs named? Give the 4 main steps.
- First letter of the genus.
- First two letters of the species.
- Letter denoting the strain.
- Number giving the class of RE.
Type II REs usually cut in a recognisable way. What is this?
They tend to leave 5’ terminal phosphates.
There are 3 main types of cut from a RE. What are they and give examples of enzymes.
- Blunt ends e.g. Bal1
- 5’ overhang e.g. Asp718
- 3’ overhang e.g. Kpn1
What enzyme can be used to digest overhangs and create blunt ends?
S1 nuclease.
What is characteristic about RE molecules and why?
They are homodimers that sit anti-parallel. Each homodimer binds to a strand of DNA. As the DNA sequence is palindromic the homodimers are aligned to mimic this.
What is a homodimer?
A molecule with 2 identical sub-units.
Where does the energy for cutting come from in RE reactions?
The energy is ‘free energy’ - it is provided by hydrolysis of the sugar-phosphate backbone of the DNA strand.
What are isoschizomers?
Different REs (often from different organisms) that have the same recognition site.
How do you calculate how often an RE cuts? Give 4 steps.
- There are 4 possible bases
- Identify the number of bases in the recognition site.
- Multiply 4 by that number, e.g. recog. site contains 4 bases: 4x4x4x4
- Answer is how many bases one cut will occur in, e.g. 4x4x4x4=256, one cut occurs every 256bp.
What is 1 unit of enzyme?
“The amount of enzyme required to cut 1μg of Lambda DNA to completion in 1 hour at 37 degrees.”
When does ‘1 unit’ not apply to an enzyme? Give 2 reasons.
- If that enzyme cannot recognise Lambda.
2. If that enzyme has an optimum temperature other than 37 degrees.
What is a DNA polymerase?
“Enzymes that synthesise nucleic acids on a pre-existing template.”
Which DNA polymerase is most commonly used in vitro for replication?
DNA polymerase I.
What are the requirements of DNA polymerase I?
A primer, nucleotides and a single strand of DNA.
DNA polymerase I has 3 main functions. What are they?
- 5’ to 3’ polymerase for replication
- 5’ to 3’ exonuclease for repair
- 3’ to 5’ exonuclease for proof-reading
What is the Klenow fragment?
When DNA polymerase I is digested with subtilisin it produces the Klenow fragment that can solely act as a 3’ to 5’ exonuclease. ALL 5’ TO 3’ ACTIVITY IS LOST.
What do DNA ligases do?
Join DNA fragments in ligation.
Sticky ends are easier to ligate than blunt ends. Which enzyme can be used to join blunt ends?
T4 DNA ligase.
How does T4 DNA ligase work?
It forms an enzyme-AMP complex than binds to 5’ terminal phosphate.
What does T4 DNA ligase require as a cofactor?
ATP.
At what temperature can sticky ends by joined at?
12-16 degrees.
What does alkaline phosphatase do?
Prevents self-ligation by removing the 5’ terminal phosphate group. Thus T4 DNA ligase cannot join blunt ends together.
What does T4 polynucleotide kinase do?
Adds a 5’ terminal phosphate, reversing the actions of alkaline phosphatase.
What is the function of DNA ligase in nature?
To seal ‘nicks’ on newly synthesised DNA, repairs breakages between bases on a single strand in the double-stranded molecule.
At what temperature does blunt ligation occur?
Room temperature.
In what process will you find DNA dependent RNA polymerases?
Transcription.
In what process will you find DNA dependent DNA polymerases?
Replication.
In what process will you find RNA dependent DNA polymerases?
Reverse transcription by retroviruses.
From what organism does DNA polymerase I originate?
E. coli.
From what organism does Taq polymerase originate?
Thermus aquaticus.
What is Amplitaq?
A GMed version of taq polymerase that is thermostable between 37-94 degrees.
What is amplitaq used in?
PCR.
What is SEQUENASE?
The brand name for bacteriophage T7 polymerase.
What is bacteriophage T7 polymerase composed of?
It is a hybrid polymerase made from 2 sub-units: thioredoxin from the E.coli host and T7 polymerase from the bacteriophage.
What is SEQUENASE used for and why?
DNA sequencing as can synthesise very long chains.
What is SEQUENASE 2.0?
SEQUENASE lacking the 3’ to 5’ exonuclease activity, this is to make it faster.
Where does reverse transcriptase come from?
Retroviruses.
What is the problem with reverse transcriptase?
It is v. error prone.
What is reverse transcriptase used for?
Creating cDNA from RNA to build cDNA libraries.
Where does S1 nuclease originate from?
The fungus A. oryzae
What enzyme can be used to open hairpin loops in newly synthesised cDNA?
S1 nuclease.
Where does ribonuclease A come from?
Bovine pancreases.
What does deoxyribonuclease do?
Randomly cleaves ss and ds DNA.
What does deoxyribonuclease require?
Mg2+.
What are the 3 uses of deoxyribonuclease?
- Introduces random nicks in DNA for radio labelling.
- Generates random sequences for cloning.
- Analyses DNA-protein complexes in footprinting.
What is DNA footprinting?
A study of the specificity of DNA-binding proteins.
