Clone DNA
DNA cloning is the beginning for almost all genetic engineering approaches to biotechnology study and research. This is because large and multiple strands of DNA are required during genetic engineering. Several copies of a DNA strand can be made either by cloning DNA in host cells or by use of polymerase chain reaction (PCR) technique. To get many copies of DNA or a gene you must cut or isolate the DNA from its source and the paste the isolated DNA into a vector that can copy or replicate itself. Cloning DNA in Host Cells A DNA fragment with a number of genes or a single gene can be inserted into a DNA vector that can be propagated or incorporated within another cell. A DNA vector is a DNA fragment that can receive another fragment of DNA without losing its self-replication capability. A vector that contains an additional fragment of DNA is referred to as Hybrid Vector. In case a DNA fragment contains one or more genes, then it is referred to as clone DNA. Below are examples of DNA vectors used during gene or DNA cloning:
Expression vectors- These are phage or plasmid vectors that have a regulatory sequence required for translation and transcription of the clone DNA. The aim of using these vectors is to make multiple copies of a protein coded by the DNA within the host cells. Plasmid vectors- They are modified forms of circular extra-chromosomal DNA segments found in bacteria. The DNA molecules can be modified to contain marker and restriction sites that help detect the bacterial cells containing the plasmid. A good example is the bacterial artificial chromosome (BAC) which is a vector made from the naturally occurring F-plasmid found in Escherichia coli bacterium. The vector is used to clone large DNA segments. Cosmids- These are artificially prepared cloning vectors that are constructed by combining plasmid with lambda phage chromosome sections. The resulting cosmid can be placed into the phage body for transmission. The vector use plasmid genes to help direct its replication within the host cells. Lambda phage vectors- these are cloning viruses that contain the phage chromosome and inserted foreign DNA. Phage vectors are used to clone larger sequences of DNA compared to plasmid vectors.
Once the DNA fragment to be cloned has been inserted into the vector, it is inserted into the host cell which copies the cloned DNA by use of its own replication mechanisms. Examples of cell types used as hosts include:
Mammalian cells Yeast cells and Bacterial cells
Procedure
Below are the four main steps in DNA cloning using host cells:
STEP 1: Choose the DNA you want to clone and then cut it from the organism. You need restriction enzymes to isolate DNA from the organism. STEP 2: The isolated DNA piece is then inserted into a vector. The DNA ends are joined with the vector DNA through a process known as ligation. STEP 3:The hybrid vector is then introduced into the host cell often yeast or a bacterium through a process known as transformation. The host cells then copy the vector DNA together with its own cells creating several copies of the inserted DNA. STEP 4: After replication, the vector DNA is separated from the host cells. The clone DNA is then purified.
DNA fragment that has been isolated and pasted from an organism into a vector is known as recombinant DNA. In this case, DNA cloning is also referred to as recombinant DNA technology. Cloning through PCR Technique Polymerase chain reaction technique is another modern way you can make multiple copies of a certain DNA section without the need of using host cells and vectors. The template DNA or the DNA to be copied is mixed with two-twenty base pairs primers which are complementary to the end of the DNA template, nucleotides, and a version of DNA polymerase called Taq polymerase. Taq polymerase is a unique enzyme that is obtained from a thermophilic bacterium. The enzyme is stable under high temperatures. PCR technique involves re repetition of the following three steps:
Denaturation-This is a process aimed to separate the 2 nucleotide strands of a DNA molecule. Annealing- Primer annealing is a process where the primers are joined to the single stranded DNA Extension- In this process, the nucleotides is joined to the primers in 5' to 3' direction to create a double-stranded copy of the DNA of interest.
Denaturation, primer annealing and extension form one PCR cycle. Each PCR cycle takes a few minutes meaning that repeated cycles can give large amounts of a certain DNA sequence in just hours. However, the details of the nucleotide piece to be copied must be known to make the process simple. The technique is simple and fast but it is sensitive to little contamination. Isolating a Clone DNA DNA cloning is undertaken for the purpose of getting the clone of a certain DNA sequence of interest. After you have cloned your DNA of interest, the next step is separating it from other DNA in the library. In order to separate, a DNA segment that shows homology to the DNA of interest is used as a probe DNA. For example, if a gene from a mouse has been cloned, then that clone can be used to find any homologous human clone from a human genome library.
What is Clone DNA used for? Cloning of DNA is aimed at creating multiple copies of a gene or piece of DNA. The cloned DNA can be used for:
Making large concentrations of a certain protein coded by the gene Investigating the characteristics of genes such as their size, tissue distribution and expression. Looking at how mutations can affect the function of the gene. Working out the gene function.
The Bottom Line The term cloning can also be used to describe other lab techniques such as: Therapeutic cloning- This process involves creation of multiple copies of a cell to help treat a certain disease. Reproductive cloning- It is a process of creating a genetically identical copy of a certain organism.