Flashcards in In Vivo cloning Deck (26):
In vivo gene cloning
Isolation and amplification of an individual gene sequence by insertion of that sequence into an organism where it can be replicated.
The product of joining together DNA from different sources
Uses for DNA cloning
- Genes can be isolated and identified
- Control sequences of DNA can be identified and analyzed
- Protein/enzyme/RNA function can be investigated
- Mutations can be identified eg: gene defeats related to specific diseases
- Organisms can be 'engineered' for specific purposes eg: insulin production, insect resistance.
Restriction enzymes (RE)
- Prevent invasion of foreign DNA (such as viral DNA) by cutting them.
- they recognize a specific DNA sequence (between 4-12bp) which is two fold symmetry and cut both DNA strands
- Depending on the enzyme they can either create staggered ends or blunt ends.
-only found in prokaryotes
Attach foreign DNA from the ends, breaking them down bit by bit.
There are ubiquitous (found everywhere)
A method of protection by bacteria to prevent RE's from damaging own DNA.
The methylase enzyme adds a methyl group to DNA at specific sites to protect the site from restriction endonuclease cleavage.
This aids in the identification of foreign DNA to its own (foreign DNA lacks the methylated group)
Where a restriction enzyme cuts the DNA, Ligase joins two DNA strands together.
A small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes
- Must contain a replicon (origin of replication) that enables it to replicate in host cells
- several marker genes (used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA)
- Unique cleavage sites
- For expression must contain control elements, such as promoters, terminators, ribsosome binding sites
Types of vectors
- Yeast Artificial Chromosomes (YACs)
- Bacterial Artificial Chromosomes (BACs)
• Naturally occurring extrachromosomal DNA
• Plasmids are circular dsDNA
• Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
• Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends of plasmid.
• can replicate independently of the host cell.
• Size ranges from a few kb to near 100kb
Cloning a gene in a bacterial plasmid
1) Vector (Bacteria containing ampicillin resistance and the human DNA containing gene of interest are cut using the same restriction enzymes.
2) Mixing the two they join by base pairing with some now containing the gene of interest
3) DNA ligase is used to covalently bond the DNA
4) plasmid is plasmid into lacZ bacteria via transformation
5) cells are cloned on a medium containing ampicillin and X-gal
6) identify clones with recombinant plasmid by the ability to grow in ampicillin and their white colour.
Why is the gene of interest inserted into the LacZ gene
Adding them into this gene makes it non functional so the bacteria is unable to breakdown lactose - this allows the plasmids containing the gene of interest to be identified. As the functional LacZ gene turns colonies blue in the presence of X-gal.
DNA can be inserted into a Cell by:
- protoplast fusion
- gene gun
Three conformations of plasmid DNA
- supercoiled: often referred to as covalently closed circular DNA, ccc
- open-circular (oc),
Which conformation runs fastest?
- Supercoiled - due to its compact natures suffers less friction through agarose
- Linear is second as it travels end first
Expressing eukaryotic genes in bacteria
- Eukaryotic DNA contains introns (intervening sequences) and exons (expressed or translated sequences), so to express the eukaryotic gene introns are edited out after mRNA transcription
Disadvantages of using bacterial host cells
May not be able to use the information in a eukaryotic gene due to the use of different enzymes.
Unable to make the post-translational modifications required to produce some eukaryotic proteins
Problem: Eukaryotic genes of interest may be two large to clone easily because they contain long noncoding regions (introns), which prevent correct expression of the gene by bacterial cells, which lack RNA-splicing machinery.
Solution: Scientists can make artificial eukaryotic genes that lack introns.
Artificial chromosomes, which combine the
essentials of a eukaryotic chromosome with foreign DNA, can carry much more DNA than plasmid vectors, thereby enabling very long pieces of DNA to be cloned.
- Plasmid vectors that contain a bacteriophage lamda cos site
- The cos site results in efficient packaging of lamda DNA into virus particles
- So, with the cos site, larger DNA inserts
are possible (up to ~40 kb)
Cosmid cloning Vectors
• Fragments from 30 to 46 kb
• Cosmids combine essential elements of a
plasmid and Lambda systems.
• Cosmids are extracted from bacteria and
mixed with restriction endonucleases.
• Cleaved cosmids are mixed with foreign
DNA that has been cleaved with the same
• Recombinant cosmids are packaged into
• Recombinant cosmid is injected into the
bacterial cell where the r cosmid arranges
into a circle and replicates as a plasmid
- Yeast Artifical Chromosome
- Artifically produced mini chromosome
- They contain a yeast centromere, two yeast teleomeres, bacterial origin of replications and marker genes
- Can accomodate large inserts (1,000 to 2,000kb)
Purpose of YACs
YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.
- A genomic library is a collection of many bacterial or phage clones, each containing copies of a particular DNA segment from a foreign genome.
- However some genes may be divided up between two or more genes.
Phage Cloning Vectors
•Fragments up to 23 kb can be may be accommodated by a phage vector
•Lambda is most common phage
•60% of the genome is needed for lyticpathway.
•Segments of the Lambda DNA is removed and a stuffer fragment is put in.
•The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector.