Week 4 (genetic Engineerig And Cloing) Flashcards
To work with a gene, it must be:
- pure
- homogeneous
- available in sufficient quantity
What is gene cloning?
Cloning is making many identical copies (of the same gene)- The amplification of a particular DNA sequence.
The term also describes the isolation of a particular stretch of
DNA from the rest of the cells genome
1 bacterial colony=
1 clone
What are the properties of a good vector?
- Be independent of the host chromosome
- Be copied by the cell
- Be maintained throughout the population of cells (every cell contains copies of the vector)
- Allow selection for cells containing the vector (as opposed to those that do not)
- It is beneficial if there is a secondary selection system which allows identification of cells containing recombinant vector
What criteria must the vector meet to have beneficial properties?
(1+2) Vectors must have an origin of replication
(3) Vectors carry a defined copy number
(3+4) Vectors carry a selectable marker e.g. antibiotic resistance
(5) vectors carry an additional selectable marker, which is inactivated by the insertion of a foreign gene
How is the vector cut?
- DNA can be cut at specific sequences by enzymes called restriction endonuclease to create linear DNA molecules
- The vectors are engineered to have just one recognition site for each group of restriction enzymes
- These recognition sites are grouped close together at one location in the plasmid (this will allow for secondary selection later on)
- Creation of single stranded sticky ends with a 5’ overhang (GATC)
- DNA ligase repeals phosphodiester bonds
- Sequences of the sticky ends of the gene complement the sequence of the plasmids sticky ends
- Transformation: puts plasmid DNA inside a cell using ice cold CaCl2 which is then heat shocked(this process is very inefficient- <1 in 10,000)
- Vector replication in which multiple copies of the same vector are made inside the cell according to the copy number encoded on the plasmid
- Selection/ cell multiplication so only cells contains the plasmid can grows
Give an example of a restriction site
BamHI
Explain secondary selection in the process of cloning?
- The restriction sites are located within a non-essential gene that produces an observable phenotype e.g betagalactosidase, which makes a blue colour
- if the plasmid re-ligands to itself in the absence of the gene, the beta galaxtosidase gene is reformed and the colony will be blue
- if the gene is inserted into the restriction site, the betagalactosidase gene (restriction site) will be disrupted and the cell will be white
Where is the restriction site cleaved by endonucleases found?
In the betagalactosidase (blue) gene not the antibiotic resistance gene
What is self ligation?
Reformation of the original plasmid without incorporating foreign DNA
What are some uses of gene cloning?
Expression- the gene product may be therapeutic, or commercially useful
Investigation- gene sequence
Manipulation- mutation of certain bases to examine how the encoded protein works
How is the bacterium’s own DNA protected from cleavage?
By methylation
Why are restriction nucleases so useful?
The enzyme will always cut a particular DNA molecule at the same sites therefore for a given sample of DNA, a particular restriction nuclease will reliably generate the same set of DNA fragments
What are plasmid vectors?
Small, circular molecules of double stranded DNA derived from plasmids that occur naturally in bacteria cells
How can the clone DNA fragment be readily recovered?
By cutting it out of the plasmid DNA with the same restriction endonuclease that was used to insert it and then separating it from the plasmid by gel electrophoresis
Why is the bacterial artificial chromosome, BAC, (an engineered derivative of the F plasmid) particularly useful?
- they are kept in low numbers therefore they can stably maintain very long DNA sequences
- with only a few BACs per bacterium, it is less likely that the cloned DNA fragments will become scrambled by recombination with sequences carried on other copies of the plasmid
- ability to accept larger DNA inserts
What is a DNA library?
The collection of cloned plasmid molecules
What is a genomic library?
DNA fragments derived directly from chromosomal DNA of organisms of interest, the resulting collection is a genomic library and will represent the entire genome of that organism.
The genomic library consists of a set of bacteria each carrying a different fragment of human DNA*
How is a cDNA clone made?
- DNA sequences that are transcribed into mRNA and thus correspond to preteen coding genes are selected
- This is done by extracting the mRNA from cells and then making a DNA copy of each mRNA molecule present
- The copying reaction is catalysed by reverse transcriptase enzymes which synthesises a complementary DNA chain on an mRNA template
- The single stranded cDNA molecules are converted by DNA polymerse into double stranded cDNA molecules and these molecules are inserted into a plasmid/vector and cloned
- Each clone obtained in this way is called a cDNA clone
An entire collection of clones derived from one mRNA preparation constitutes a cDNA library*
What is the difference between genomic and cDNA clones?
- Genomic clones represent a random sample of all of the DNA sequence in an organism (both coding and non coding) and are the same regardless of cell type used to prepare them
- cDNA clones contain only those regions of the genome that have been transcribed into mRNA. Because the cells of different tissues produce distinct sets of mRNA molecules a distinct DNA library is obtained for each type of cell used to prepare the library
How was the nucleotide sequence of the human genome determined?
- It was broken 100, 000 nucleotide pair pieces, each of which was inserted into a BAC plasmids and amplified in E. Coli
- The resulting genomic library consisted of tens of thousands of bacterial colonies, each containing a different human DNA insert
- The nucleotide sequence of each insert was determined separately and the entire genome was stitched together from the pieces
What is the most important advantage of cDNA clones over genomic clones?
- They contain the uninterrupted coding sequence of a gene
- when the aim of the cloning is to produce the protein in large quantities by expressing the cloned gene in a bacterial or yeast cell, it is more preferable to start with cDNA
What are the two major reasons for cloning?
To make biological products e.g human growth hormone
To enable academic study
What are biological products of cloning?
- e.g human growth hormone (originally extracted from cadavers but lead to some cases of CJD- Now produced in bacteria
- insulin (originally extracted from pigs/cows - some immune problems so now produced in bacteria)
- erythropoietin (used to treat anaemia, now available commercially prior to recombinant DNA technology) produced in CHO (Chinese Hamster Ovaries)
