Abi Starr
Chapter 16.2: In vivo gene cloning – the use of vectors
After the DNA fragments have been obtained, the next stage is cloning them so there are enough of
them for medical or commercial use. This can be done in two ways:
In vivo – by transferring the fragments to a host cell using a vector
In vitro – using the polymerase chain reactions
Importance of ‘sticky ends’
The sequences of DNA that are cut by the restriction endonuclease enzymes are called recognition
sites. If the recognition site is cut in a staggered fashion, the cut ends of the DNA double strand are
left with a single strand which is a few nucleotide bases long. The nucleotides on the single strand at
one side of the cut are complementary to those at the other side because they were previously
paired together.
If the same restriction endonuclease is used to cut DNA, then all the fragments produced will have
ends that are complementary to one another. This means that the single-stranded end of any one
fragment can be joined to the single stranded end of any other fragment. Their ends are ‘sticky’.
Once the complementary bases of two ‘sticky ends’ have paired up, DNA ligase is used to reform the
sugar-phosphate backbone.
Sticky ends are important because, if the same restriction endonuclease enzyme is used, we can
combine the DNA of one organism with that of any other organism.
Insertion of DNA fragments into a vector
Once a DNA fragment has been cut from the rest of the DNA, the next thing to do is insert it into a
carrying unit, in this case, a vector. This vector is used to transport the DNA into the host cell. There
are different types of vector, but the most commonly used is the plasmid. Plasmids are circular DNA
found in bacteria, which are separate from the main bacterial DNA. Plasmids almost always contain
genes for antibiotic resistance, and restriction endonucleases are used are used at one of these
antibiotic-resistance genes to break the plasmid loop.
The restriction endonuclease used is the same as the one that cut out the original DNA fragment.
This ensures that the ‘sticky ends’ of the opened-up plasmid are complementary to the ‘sticky ends’
of the DNA fragment. When the DNA fragments are mixed with the opened up plasmids, they may
become incorporated into them. Where they are incorporated, the join is made permanent using
DNA ligase. These plasmids now have recombinant DNA.
Page 1 of 3
Chapter 16.2: In vivo gene cloning – the use of vectors
After the DNA fragments have been obtained, the next stage is cloning them so there are enough of
them for medical or commercial use. This can be done in two ways:
In vivo – by transferring the fragments to a host cell using a vector
In vitro – using the polymerase chain reactions
Importance of ‘sticky ends’
The sequences of DNA that are cut by the restriction endonuclease enzymes are called recognition
sites. If the recognition site is cut in a staggered fashion, the cut ends of the DNA double strand are
left with a single strand which is a few nucleotide bases long. The nucleotides on the single strand at
one side of the cut are complementary to those at the other side because they were previously
paired together.
If the same restriction endonuclease is used to cut DNA, then all the fragments produced will have
ends that are complementary to one another. This means that the single-stranded end of any one
fragment can be joined to the single stranded end of any other fragment. Their ends are ‘sticky’.
Once the complementary bases of two ‘sticky ends’ have paired up, DNA ligase is used to reform the
sugar-phosphate backbone.
Sticky ends are important because, if the same restriction endonuclease enzyme is used, we can
combine the DNA of one organism with that of any other organism.
Insertion of DNA fragments into a vector
Once a DNA fragment has been cut from the rest of the DNA, the next thing to do is insert it into a
carrying unit, in this case, a vector. This vector is used to transport the DNA into the host cell. There
are different types of vector, but the most commonly used is the plasmid. Plasmids are circular DNA
found in bacteria, which are separate from the main bacterial DNA. Plasmids almost always contain
genes for antibiotic resistance, and restriction endonucleases are used are used at one of these
antibiotic-resistance genes to break the plasmid loop.
The restriction endonuclease used is the same as the one that cut out the original DNA fragment.
This ensures that the ‘sticky ends’ of the opened-up plasmid are complementary to the ‘sticky ends’
of the DNA fragment. When the DNA fragments are mixed with the opened up plasmids, they may
become incorporated into them. Where they are incorporated, the join is made permanent using
DNA ligase. These plasmids now have recombinant DNA.
Page 1 of 3
