Gene Expression 4 - Chapter Four- Gene Cloning
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What is a Clone?
- Two or more identical cells or organisms.
- Identical Twins are natural clones.
What do Restriction Endonucleases do?
Restriction Endonucleases cut DNA at or near specific base sequences.
A restriction endonuclease is taken from S. Pneumoniae.
The enzyme is derived from strain a, and is the third of five endonucleases.
What would be the name of this Restriction Endonuclease?
- The first letter (S) comes from the genus (Streptococcus), the following two from the species (Pneumoniae).The A comes from the particular strain it was derived from.
- The III describes which particular Restriction Endonuclease (if there are multiple).
Which organism might we use to derive the Restriction Endonuclease SmaAII from?
A) Ebolavirus Zaire
B) Streptococcus Rabidae
C) Escherachia Coli
D) Serratia Marcessens
D) Serratia Marcessens
Which bacterium does HindII come from?
A) Haemophilus Influenzae
B) Haemophilus Islitidae
C) Haemophius Anarchedae
A) Haemophilus Influenzae
What is the specific sequence that HindII recognizes?
- *(The "||" indicates the cleavage point).
Different Restriction Endonucleases will recognize different length sequences. Will a Restriction Endonuclease requiring a longer sequence cut more or less frequently than one requiring a short sequence?
- A Restriction Endonuclease that recognizes a long sequence will cut less frequently than one requiring a short sequence.
- e.g A Restriction Endonuclease recognizing six bases will cut the DNA once in approximately 46 bases, whereas one recognizing four will cut one in approximately 44.
What is a Rare Cutter?
A restriction enzyme that cuts only rarely because it's recognition site is uncommon.
Give one example of a Rare Cutter.
NotI (8 Base recognition sequence).
The terms Heteroschizomers and Neoschizomers are used interchangeably. What do they mean?
Neo-/Heteroschizomers refer to two Restriction Endonucleases that recognize exactly the same sequence but cleave it at different places.
Two different Restriction Endonucleases that cut at the same place on the same site of DNA are known as?
What is being shown in the picture?
- Most noteably Sticky Ends as a result of asymmetric cleavage.
- Also, the sequence is palindromic.
What feature of Restriction Endonuclease recognition sites make it possible for the enzyme to cut asymmetrically?
The recognition site is palindromic, so often the Restriction Endonuclease is recognizing the same bases, only in reverse.
What is the role of Methylase?
Methylase methylates native DNA to stop Restriction Endonucleases from cleaving it.
What is the R-M System?
R-M Stands for Restriction Methylation. This is the system by which native DNA is methylated to stop Restriction Endonucleases from cleaving it.
Why might Restriction Endonucleases be useful if not for cutting up Native DNA?
Restriction Endonucleases serve to cut up foreign DNA to stop infection from other organisms. Most notably Viruses.
How come newly replicated DNA is not cleaved by Restriction Endonucleases?
What is a Vector?
DNA (often a plasmid, but not always) that is used as a host for gene cloning experiments.
Why might it be necessary to use a Vector?
Often, DNA being examined does not have an origin of replication. Therefore, a vector is a useful way of obtaining more DNA relatively easily, as the host DNA will have all necessary genes and sequences.
There are two main types of vector. What are they?
What is the first stage in Gene cloning?
- Insert the DNA into a bacteria by Transformation.
- This is often done by either using Calcium salts to give the cells leaky membranes (Increasing competency) or using Electroporation.
What the pUC series? Why is it especially useful?
- The pUC series is a series of Vector plasmids that can be used in gene cloning.
- The pUC vector plasmid has a resistance to ampicillin to allow for screening.
- pUC plasmids have multiple Restriction Endonuclease recognition sequences in a small space. This is known as a Multiple Cloning Site.
What does "In Vitro" mean?
- Doing something "In Vitro" means doing it outside of the organism.
- (In a test tube).
What does "In Vivo" mean?
Doing something "In Vivo" means doing it outside of the organism.
The MCS in pCU is on the lacZ' gene. What does this gene code for?
lacZ' codes for the Amino Terminal portion of Beta-Galactase. (The Carboxyl portion is found in the host genome).
How might you screen a culture of potentially recombinant bacteria that have been transformed with the pUC plasmid?
- Due to the location of the MCS in the lacZ' gene, any recombinant bacteria will be white (as oppose to blue) as an indirect result of the recombinant DNA making the gene faulty.
- However, any bacteria that do not have the plasmid may also be white. Therefore, the white cultures can be plated on Ampicillin. Those that survive are recombinant.
Why do the non-recombinant (sort of) cells containing the pUC plasmid turn blue?
- The lacZ' gene allows for the completion of the beta-galactosidase protein that breaks down lactose and similar substrates (X-gal).
- When the culture is plated on media containing X-gal, the X-gal will break down into galactose and a blue dye, giving the colony colour.
Using beta-galactosidase, what are the products of reactions with,
- Lactose + Beta Galactosidase --> Galactose + Glucose.
- X-Gal + Beta Galactosidase --> Galactose + Blue Dye.
Due to the semiconservative nature of DNA, although the new strand my not be methylated, the precusor strand will be. Restriction endonucleases need to be able to interact with both strands to cleave, therefore one methylated strand will prevent the whole thing.
Using one Restriction Endonuclease, a DNA Ligase and two related but different plasmids. How might you go about making a recombinant?
- Combine the Restriction Endonuclease with the two plasmids, so they cleave them at the same sequence (assuming they share one).
- The result will be two linear DNA molecules with complimentary sticky ends.
- Allow the two plasmids to anneal together (sticky ends join up).
- Use DNA ligase to complete the phospodiester backbone.
- Finally, screen for recombinants (as this process is not 100% effective).
When making recombinants for gene cloning, what are three possible outcomes for the final product?
- Cells don't take up the DNA
- Cells take up the plasmid, without the added DNA.
- Everything is successful and ready to go
What are some advantages of gene cloning?
- Allows for creation of large quantities of DNA
- Gives information on Gene function
- Allows manipulation of gene sequences
What are the two key enzymes used in DNA cloning?
- Restriction Endonucleases
- DNA Ligase
DNA is inserted into Bacteria by Transformation. What is the term used to describe the similar process in Eukaryotes?
Would this sequence still be Palindromic if another base was added? Explain.
No, palindromes must always be a multiple of two.
Why might you add Alkaline Phosphatase to the Vector Plasmid when cloning DNA?
Alkaline Phosphatase will remove phosphate groups from the plasmid 5' ends and therefore prevent self ligation.
What is Directional cloning? How might you go about doing it?
- Directional cloning is the insertion of DNA into the vector plasmid in only one orientation.
- Directional cloning can be achieved by using two Restriction Endonucleases as oppose to just one, to give two different sticky end sequences.
What are the advantages of Phage Gene cloning?
- Phage cloning is more effective than Plasmid Gene cloning because Phages are more efficient at inserting their DNA.
- Phages have larger genomes than the Plasmid vectors, therefore larger inserts can be added.
Replacement Vectors are phages that have had some of their genome replaced by foreign DNA. What is another name for this type of Vector?
Why might it be useful to clone a large segment of DNA with one vector organism?
The cloning of large genomes is useful for sequencing of Genomic Libraries.
What are the requirements of the host phage for foreign DNA?
The DNA must be a minimum of 12kb to be accepted, but no more than 20kb (to fit the head).
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