Gene Cloning: Facts, Information, and Resources
What is Gene Cloning?
Gene Cloning is the term given to the process of in vitro multiplication of a DNA fragment after obtaining it from any source. This DNA fragment can be a gene sequence coding for some protein or promoter sites that are used as signals for the start of transcription of the proteins. These can be obtained from the genome of any plant or animal, or they can also be man- made, a few oligonucleotides in length.
Methods of Gene Cloning
Gene cloning can be done by two main methods:
- By using vectors
- By PCR
By Using Vectors
Vectors are agents used for transferring the gene or the DNA sequence into a host cell and replicating this DNA sequence inside the host cell. The most commonly used vectors are derived from bacteria and viruses and they have uses in various techniques in molecular biology processes. A vector must have certain specific qualities to be able to be used as a vector. They are as follows:
- A vector must be able to independently replicate within the host cell and produce large copy number.
- It must be able to transfer itself to new daughter cells so that it can produce more copies of itself.
- It must not be very large in size and must be smaller than 10kb. Smaller molecules are more stable and can be easily manipulated for formation of recombinant DNA molecules.
Two major types of molecules that fulfill these criterions are plasmids and phage DNA .
Plasmids
Plasmids are extra-chromosomal, circular DNA molecules found in some bacterial cells, providing some specific characters like antibiotic resistance to that bacterial cell. Almost all plasmids have genes for origin of replication, the site where the replication of DNA strands start, allowing it to replicate independent of the bacterial chromosome. Factors affecting the selection of the plasmid as vector are:
1) The size of the plasmids (plasmids to be used as vectors must be small and not more than 10 kb).
2)There must be multiple copies of the plasmid present inside single host cell. (This is known as copy number) the higher the copy number, the more copies of the desired DNA fragment will be produced from each cell.
Plasmids can be put into two main groups (i) conjugative and (ii) non-conjugative.
There are five major types of plasmids
1- Fertility or F plasmids
2- Resistance or R plasmids
3- Col-plasmids
4- Degradative plasmids
5- Virulence or vir- plasmids
Bacteriophages
The viruses that specifically infect bacterial cells are known as bacteriophage. These viruses attaches themselves on the bacterium, injects its DNA in the bacterial cell, replicates its own DNA inside the bacterium, synthesizes its capsid and all other components and then assembles all the parts together and finally ruptures the bacterial cell and gets released. The phage cycles can be of two types
1-Lytic cycle: Phages that complete the infection cycle in a very short time.
2-Lysogenic cycle: Phages that keep their DNA integrated in the host cell DNA (called as prophage) and after many replication cycles of the host reverts to the lytic phase and infects the bacterial cells.
Cloning By PCR
PCR or Polymerase Chain Reaction is a method in which the amplification of Short DNA sequences is achieved in a thermal cycler using a set of chemicals in a few steps:
(i) Denaturation
In this stage the double stranded DNA molecule that is to be amplified, is denatured by exposing it to very high temperature 940C .
(ii) Annealing
Temperature is cooled down little to let the primers get attached at the ends of the denatured dsDNA .
(iii)synthesis
In this step the Taq polymerase, a DNA polymerase that has the ability to replicate at higher temperatures efficiently, is used to replicate these strands of DNA that have the primers attached on their ends.
These steps are repeated up to 20 to 25 times to give a very high density of the DNA that was to be amplified.
Limitations of PCR
Only short sequences can be amplified by using this technique.
Only gene sequences that have already been studied can be amplified with purity by this method.
Gene expressions cannot be studied by this method.
Uses of PCR
PCR is used to detect genetic diseases, DNA fingerprinting for forensic medicine and for establishing paternity. DNA fingerprinting of endangered animals is also done to keep a track on their growth records. PCR is also used for studying human evolution.
Resources About Cloning
SDSU.edu’s information about gene cloning.
Ornl.gov questions about cloning such as:
What is cloning?
Are there different types of cloning?
How can cloning technologies be used?
What animals have been cloned?
Can organs be cloned for use in transplants?
What are the risks of cloning?
Should humans be cloned?
DNA Cloning and Sequencing.
The Basics of cloning
Genome.gov questions and answers about genetic cloning such as:
What is cloning?
Do clones ever occur naturally?
What are the types of artificial cloning?
What sort of cloning research is going on at NHGRI?
How are genes cloned?
How are animals cloned?
What animals have been cloned?
Have humans been cloned?
Do cloned animals always look identical?
What are the potential applications of cloning animals?
What are the potential drawbacks of cloning animals?
What is therapeutic cloning?
What are the potential applications of therapeutic cloning?
What are the potential drawbacks of therapeutic cloning?
What are some of the ethical issues related to cloning?
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