12-27-2013, 09:17 PM
Cloning of a gene for expression purpose (in bacterial, yeast, mammalian or other systems) involves many steps. Depending up on the information available on the gene of interest, various methods can be used to isolate and amplify it.
Steps for the cloning of a gene
1. Getting the Gene of interest:
Firstly you must get the DNA and that you quantity in sufficient quantities to clone, you need to use following method depending upon the availability of starting materials.
a) DNA as starting material: (From genomic DNA or piece of DNA),
If you had a very small quantity of the DNA and knew enough of the sequence to make primers you can use PCR to amplify it.
b) RNA as starting material: (eg mRNA)
You would need to use reverse transcriptase to 'reverse transcribe' it into DNA fallowed by PCR amplification to get the product.
c) Protein as starting material:
If you know the sequence of protein then you need software to reverse translate it to get the gene sequence. You can bias it depending upon the expression system.
2. Vector selection for expression of gene:
Most of the time gene is cloned for expression purposes and depending of the requirement/application you can select vector. A vector is circular DNA (Modified Plasmid) that replicates in a bacterium or yeast system so it has origin of replication for that system.
So, you can see, a gene inserted into a plasmid will be replicated many of times in single bacteria. And it is possible to grow millions of bacteria in a 1 litre flask. So this is a good way to make lots of copies of a gene of interest.
These vectors are now a day’s commercially available which will have restriction sites for cloning and upstream regulatory sequence which will help for better expression of gene.
3. Cloning of DNA:
Next you will use restriction enzymes to cut out the DNA/gene you want from the amplified product. You use the same restriction enzymes to cut the vector also, so that the ends will be compatible. This means that if you will use a "sticky ends" restriction enzymes (Available in market), then the DNA sequences of the trailing bits will be compatible and will want to stick to each other. This makes it much easier to do the cloning.
Once restriction digestion is done you can purify the specific DNA from Agarose gel using Gel electrophoresis. These fragment (Vector + Gene/DNA) can be ligated using a Ligase enzyme (Available commercially)
Once the ligation is done, it can be transformed to Bacteria strains (E coli) where is will be amplified while growing on Agar plate. Here you will get many colonies of E coli which will contain clones of DNA in vector. These clones can be further grown in culture media and DNA can be isolated from here.
Sometime you will get false clone which can be further eliminated by screening using PCR or restriction digestion. Once you get the positive clone they can be used for expression in bacterial or homologous systems.
Steps for the cloning of a gene
1. Getting the Gene of interest:
Firstly you must get the DNA and that you quantity in sufficient quantities to clone, you need to use following method depending upon the availability of starting materials.
a) DNA as starting material: (From genomic DNA or piece of DNA),
If you had a very small quantity of the DNA and knew enough of the sequence to make primers you can use PCR to amplify it.
b) RNA as starting material: (eg mRNA)
You would need to use reverse transcriptase to 'reverse transcribe' it into DNA fallowed by PCR amplification to get the product.
c) Protein as starting material:
If you know the sequence of protein then you need software to reverse translate it to get the gene sequence. You can bias it depending upon the expression system.
2. Vector selection for expression of gene:
Most of the time gene is cloned for expression purposes and depending of the requirement/application you can select vector. A vector is circular DNA (Modified Plasmid) that replicates in a bacterium or yeast system so it has origin of replication for that system.
So, you can see, a gene inserted into a plasmid will be replicated many of times in single bacteria. And it is possible to grow millions of bacteria in a 1 litre flask. So this is a good way to make lots of copies of a gene of interest.
These vectors are now a day’s commercially available which will have restriction sites for cloning and upstream regulatory sequence which will help for better expression of gene.
3. Cloning of DNA:
Next you will use restriction enzymes to cut out the DNA/gene you want from the amplified product. You use the same restriction enzymes to cut the vector also, so that the ends will be compatible. This means that if you will use a "sticky ends" restriction enzymes (Available in market), then the DNA sequences of the trailing bits will be compatible and will want to stick to each other. This makes it much easier to do the cloning.
Once restriction digestion is done you can purify the specific DNA from Agarose gel using Gel electrophoresis. These fragment (Vector + Gene/DNA) can be ligated using a Ligase enzyme (Available commercially)
Once the ligation is done, it can be transformed to Bacteria strains (E coli) where is will be amplified while growing on Agar plate. Here you will get many colonies of E coli which will contain clones of DNA in vector. These clones can be further grown in culture media and DNA can be isolated from here.
Sometime you will get false clone which can be further eliminated by screening using PCR or restriction digestion. Once you get the positive clone they can be used for expression in bacterial or homologous systems.
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