Cloning Flashcards
Define clones
A collection of identical organisms that are derived from a single ancestor
What are molecular cloning techniques also called
Genetic engineering or recombinant DNA technology
What is the main idea behind cloning
To insert a DNA segment into an autonomously replicating DNA molecule, called a cloning vector or vehicle, which will then replicate the DNA segment along with it
Give 2 examples of suitable host organisms for cloning
E-coli and yeast
What is the result of cloning
To produce large amounts of the inserted DNA segment, if the DNA is inserted near the transcription and translation control sequences the vector might even produce the RNA and proteins for the corresponding gene
Define an endonuclease
An enzyme that hydrolytically cleaves polynucleotides at internal sites
Define a restriction endonuclease
An enzyme that recognizes a specific DNA sequence that is 4-8 bases and cleaves it from the DNA double helix on both strands
How did researches discover host-specific modification
They realized that when a bacteriophage propagates a certain strain of bacterium efficiently eg. E-coli K12, they have a low rate of infection on a related bacterial strain eg. E-coli B. However, when the few viral progeny from the related bacterial strain was introduced to a new host it propagated efficiently, but only poorly in the original host. This shows that the new host must have modified the bacteriophages in some way
What is the molecular basis of host-specific modification
It results from a restriction-modification system in the host, that consists of restriction endonuclease and DNA methyltransferase
Define DNA methyltransferase
An enzyme that methylates a specific base on the same DNA sequence that is recognized by the restriction enzyme
Why doesn’t a restriction enzyme cleave a methylated DNA
The methyl group protects the DNA from being degraded
How does the restriction-modification system protect against foreign DNA
They’re cleaved by restriction endonuclease and further degraded by bacterial exonuclease
What occurs if the foreign DNA becomes methylated
The restriction endonucleases and bacterial exonucleases cannot cleave it, so it can reproduce in the bacterium. But, the progeny is not methylated the same way that allows it to propagate in the host
Name the 4 types of restriction endonucleases
Type I, II, III and IV
What do type I and III restriction endonucleases have in common
They both carry endonuclease and DNA methyltransferase activity on one protein molecule
List each restriction endonuclease and their function
Type I- cleaves the DNA at a random site least 1000 bp from the recognition site
Type II- is a separate entity from its DNA methyltransferase, cleaves at specific sites on DNA within or near the recognition site
Type III- cleaves DNA at 24-26 bp from the recognition sequence
Type IV- cleaves methylated DNA
What is the most common recognition sites that type II restriction endonucleases recognize
Palindromes(read the same backwards and forward)
What are the 2 ways that type II restriction enzymes catalyze the cleavage of a DNA sequence
They either catalyze cleavage of the two DNA strands at positions that are symmetrically staggered about the center of the palindromic recognition sequence which creates sticky ends (complementary single stranded ends that are 1 to 4 nucleotides in length) or they catalyzed cleavage at the center or the palindromic sequence which creates blunt ends. Either restriction fragments with sticky ends are created or blunt ends
What is produced after a DNA sequence is cleaved by a restriction endonuclease
A well defined DNA fragment
What method can be used to separate the DNA fragments and why
Gel electrophoresis can be used to separate the DNA fragments because it separates them according to size
Explain the basic idea behind gel electrophoresis
Charged molecules are placed into a thin slab of agrose gel on one end and there is an electric field that is applied. This causes the smaller molecules to move faster and therefore be closer to the bottom and the larger molecules will be at the top
Explain the 2 methods used to separate the DNA fragments in to single strands
Melt the DNA fragments and then subject it to gel electrophoresis or use density gradient ultrafugation in an alkaline solution (to denature DNA)
What do genetic polymorphisms bring about in species
Individuality
How do genetic polymorphisms bring about individuality in different species
Since individuals have genetic differences, this can eliminate or create different restriction sites. Restriction endonucleases fragment DNA on different chromosomes, which creates restriction fragment length polymorphysims (RFLP, fragments with different lengths can be used to identify individuals)
Provide 3 example of vectors that are used in genetic engineering
Plasmids, viruses and artificial chromosomes
What are plasmids
Double-stranded circular DNA that is 1-200kb long and contains the genetic machinery for their autonomous replication, eg. Origin of replication( ori- site where replication is initiated) in a bacterial or yeast host.
What are some of the benefits of plasmids (from the hosts POV)
They provide resistance to antibiotics which the host lacks. They are part of the reason antibiotic-resistant pathogens are more common now
Discuss the 2 methods of replication used in plasmids
Some plasmids use stringent control to replicate this is when one or a few copies are present per cell and it replicates once every cell division. Most plasmids use relaxed control, they are present in 10-700 copies per cell.
What happens to replication in plasmids if the bacterial protein synthesis is inhibited by an antibiotic
These antibiotics will cause cell division in the bacterium to stop, but replication in the plasmid will continue until there are around 2000-3000 copied in the bacterium
How are plasmids created
Genetic engineering techniques
What is the purpose of a plasmid
They are used as vectors in molecular cloning
List some of the features that make plasmids perfect for use in molecular cloning
They are small, most plasmids used relaxed control to replicate therefore producing more copies, they carry genes that specify for resistance to one or more antibiotics and contain a number of conveniently located endonuclease restriction sites for the DNA that is to be cloned to be inserted.
What is a polylinker
A short segment of DNA located in the plasmid that contains a variety of endonuclease restriction sites that is not found anywhere else in the plasmid
Explain transformation in a bacterium
The host bacterium takes up the plasmid when the two are mixed together in a process that is enhanced by the presence of divalent cations,eg. Ca2+ and brief heating to around 42 degrees (increases the cell membranes permeability to the DNA).
Why can’t plasmids be used to clone DNA that is longer than 10kb
The time required for plasmid replication increases as the size of the plasmid increases. These plasmids will be lost due to random deletions
What is the length of a DNA fragment that a bacteriophage lamda can clone
Up to 16kb
Why can bacteriophage lamda be used to clone DNA that is slightly longer compared to the size of a DNA fragment cloned in a plasmid
The genome of a bacteriophage consists of 48.5kb and the central third of the genome is not necessary for phage infection on the bacterium so it can be replaced by foreign DNA that is slightly longer
How can the chimeric bacteriophage lamda DNA infect the host
The chimeric bacteriophage lamda DNA is inserted by an in vitro packaging system
What are the advantages of using bacteriophage lamda as a cloning vector
It produces large volumes of chimeric DNA and in a purified form
What is a cosmid vector and how is it made
When the DNA insert in a bacteriophage lamda contains a specific 16bp sequence known as a cos site at each end and are 36-51 kb apart, they can be taken and placed a proper distance apart on a plasimid vector using an in vitro packaging system- this creates a cosmid vector. A cosmid vector can contain an insert of up to 49kb. Cosmids have no phage genes so when it is introduced into a host via phage infection it reproduces as a plasmid
What is filamentous bacteriophage M13
A cloning vector that has single-stranded circular DNA that is contained in a protein tube composed of about 2700 helically arranged identical subunits
What determines the length of the subunits in filamentous bacteriophage M13
The length of the phage DNA being coated- insertion of the foreign DNA in a nonessential region of the M13 chromosome results in the production of longer phage particles
M13 cloning vectors cannot stably maintain DNA inserts of longer than 1kb, so why are used in cloning
They are used in production of DNA for sequence analysis- the phages directly produce the single-stranded DNA that the technique requires
What are baculoviruses
A large and diverse group of pathogenic viruses that mainly infect insects (not vertebrates= safe for lab use) and can be grown in cultures of insect cells
What in the genome of baculoviruses makes it possible for them to be cloning vectors
There is a segment of the double-stranded DNA that is not necessary in replication that can be replaced by the insert of DNA of up to 15kb that needs to be cloned
What cloning vector can you use if the DNA insert is too large for a cosmid vector
Yeast Artificial Chromosomes (YACs) or Bacterial Artificial Chromosomes (BACs)
What are YACs
A linear DNA segment that contains all the genetic machinery required to carry out replication in yeast: a replication origin(autonomously replicating sequence- ARS), centromere(the chromosomal segment that attaches to the spindle during meiosis and meiosis) and telomeres(ends of linear chromosomes that allow them to replicate)
What are BACs
They replicate in E-coli and are derived from circular plasmids that normally replicate long regions of DNA and and are maintained at a level of approximately one copy per cell. These vectors contain the minimal sequences required for autonomous replication, copy number control and the proper partitioning of the plasmid during cell division
What length of DNA inserts can YACs and BACs clone
Several hundred kb inserts
How is a DNA that is about to be cloned obtained
It is obtained as a sequence-defined fragment by the use of restriction endonucleases
What is the extra step that is needed for filamentous bacteriophage M13s DNA fragment to be cloned
Since it has single-stranded circular DNA and restriction endonucleases need double-stranded DNA , DNA polymerase I acts on the single-stranded DNA
Most restriction enzymes cleave DNA at specific palindromic sites to yield complementary ends( blunt or sticky), so how does a DNA fragment join the DNA
The restriction fragment may be inserted into a cut that was made by the same restriction endonuclease and the two complementary ends of the fragments associate under annealing conditions and are joined by DNA ligase
What is the advantage of using restriction enzymes to construct chimeric vectors
The same restriction endonuclease can be used to cleave the vector and the DNA fragment. It can also excise the foreign DNA from the vector
If the cloning vector and the DNA fragment have no common restriction sites then how can both be spliced
There is a procedure that uses terminal deoxynucleotidyl transferase (terminal transferase). This mammalian enzyme adds nucleotides to the 3’-terminal OH group of a DNA chain (does not require a template). Terminal transferase and dTTP(for example) can add around 100 poly(dT) tails to the 3’-end of the OH group of the DNA fragment to be cloned. Then the cloning vector is cleaved at a specific site and poly(dA) tails (for example) are added to the 3’-end of the OH group. The complementary tails are now annealed, any gaps that formed because of differences in lengths of the tails are filled by DNA polymerase I and joined by DNA ligase.
What is the disadvantage of using this technique
It eliminates the restriction sites that were used to create the DNA fragment and cleave the vector. It may be difficult to recover the insert from the vector.
How can we overcome this disadvantage caused by this technique
There is a technique in which a chemically synthesized palindromic “linker” that has a restriction site that matches the cloning vector is attached to both ends of the foreign DNA. The linker is attached to the foreign DNA by blunt end ligation with T4 DNA ligase and then cleaved with the appropriate restriction enzyme to yield cohesive ends for ligation to the vector
How can one select only those host organisms that have been transformed by the properly constructed vector, specifically in the case of plasmid transformation
This is usually done through a double screen using antibiotics and/or chromogenic substrates (color-producing)
Provide an example of how a host organism would be screened to test whether it has been properly transformed
The pUC18 plasmid contains the lacZ’ gene(modified Z gene), which encodes the enzyme beta-galactosidase( catalyzes the hydrolysis of a bond from the O1 of the sugar beta-D-galactose to a substituent). When bacteria is grown in the presence of X-gal( 5-bromo-4-chloro-3-indoyl-beta-D-galactoside, a colorless substance that turns blue when hydrolyzed by beta-galactosidase), specifically when E-coli is transformed by an unmodified pUC18 plasmid the colonies turn blue. But, if E-coli is transformed by pUC18 plasmid with a foreign DNA insert in its polylinker region it forms colorless colonies- this is because the DNA insert interupts the protein-encoding sequence of the lac Z’-gene and hence lacks beta-galactosidase activity. To exclude bacteria that have not taken up the plasmid an antibiotic called ampicillin is added to the growth medium. Bacteria that do not contain the plasmid are sensitive to the ampicillin, whereas bacteria containing the plasmid will grow. This is because the plasmid has a gene called amp^R that enables it to have anti-biotic resistance.
What are genes such as amp^R collectively called
Selectable markers
