27. Gene Cloning Flashcards
what is gene cloning?
copying segments of a DNA into many copies, then inserting it into another organisms. study image!
what are restrictive enzymes used for in gene cloning? how do they work?
restriction enzymes cut out a segment of a large piece of DNA (such as a genome).
cut the DNA by hydrolising sugar-phosphate bonds in the backbone
why does bacteria make restiction enymes?
how does bacteria protect their own DNA against their own restriction enyzmes?
restriction enymes in bacteria are made to protect the cell by cutting foreign DNA from other organisms eg phages
bacteria protect their own DNA from these enymes by the addition of methyl to adenines or cytosines within the sequences recognised by the enymes
what is the ‘sticky end’ that restirction enymes make in DNA?
within the restrictin site, the restirction enyme cuts the sugar phosphate backbones with some of each strand hanging over each other (refer to image) .
this creates ‘sticky ends’ to make it easier for the fragments to bind to othe DNA fragments eg a plasmid
where is the gene of interest carried in gene cloining? define what this is
its carried on a plasmid. this is DNA within the cell that is physically seperated from chromosmal DNA and replicated independatly
what is a cloning vector?
a DNA plasmid that is being used for cloning
what is a recombinant DNA?
when the same type of restriction enyme is used to cut both a plasmid and DNA containing a gene of interest, they will have compatible sticky ends and can be joined using DNA ligase which forms a recombinant DNA
how does the process of gene cloning result in a cell containing a recombinant plasmid (DNA)?
explain the process of gene cloning and what the process is called
Restriction enymes cut out the gene of interest in the bacteria cell plasmid. This makes the plasmid linear (when its usually circular)
the same restirction enyme will cut out a fragment of a foreign gene, DNA ligase combines the sticky ends to make a recombinant plasmid.
the plasmid has antibiotic gene resitance, so in incubators an antibiotic will be exposed to them, so only the bacteria that has the resistnace on the plasmid (and the GMO) will survive and reproduce. all cells will then have a recombinant plasmid through the process of transformation!!
what are the similarities between the role of enymes used in DNA technology and their natural biological roles in the cell?
restriciton enymes (2 points) and DNA ligase (1 point)
restriction enymes are used to cut up DNA by breaking the hydrogen bonds in both situations. they are both specific and cut at a restriction site.
DNA ligase are used to bind together the DNA double helix again in both situations
what are the different roles of restriciton enymes and DNA ligase in natural biological roles vs gene cloning
restriction enymes are used for protection to cut up and destroy phages coming into the bacteria cell, whereas in cloning its used to cut out genes of interest
DNA ligase is used for gene expression to put the DNA back together, whereas in gene cloning its to bind the foreign and original gene back together
what are the features of plasmids that enable them to be used as cloning vectors
plasmids have an origin of replication:
- ensures when the foreign gene is added, the vector plasmid is replictated
- (think about transformation of f1 to f2 plasmids)
plasmids tend to hold an anitbiotic resistance gene, if not then it can be inserted into the plasmid.
- used as a genetic marker to know the gene has the GMO
- so the bacteria dosent loose the plasmid
- natural selection
what are the two ways foreign genes can be obtained into the plasmid?
PCR with primers
restriction digest
