DNA Cloning

What is a DNA cloning vector?

Vectors are pieces of DNA that can have other pieces of DNA inserted into them. These vectors are what will be able to deliver DNA into host cells. There are a few different vectors that can be used such as plasmids (from bacteria), viral vectors (from viruses), cosmids (a type of hybrid plasmid), and artificial chromosomes (manmade chromosomes that contain desired features).

DNA cloning purpose

There are multiple reasons why scientists want to use DNA cloning:

• Produce large amounts of desired DNA or proteins.

• Create recombinant DNA to study how genes function.

• Study potential gene mutations and how they can alter a gene's function.

• Study the characteristics of genes.

DNA cloning can also be used in the medical field! Utilizing recombinant DNA, desired human proteins can be given to bacteria cells. Bacteria cells become able to grow these proteins which can be harvested for human medications! Two examples of these are insulin and human growth hormones.

DNA cloning steps

In order to clone DNA, there needs to be a multi-step process. The steps for DNA cloning are:

1. Isolation of desired DNA

2. Ligation

3. Transformation

4. Screening procedure

Isolation of desired DNA

In order to use the gene or DNA of interest, it must be isolated from the rest of the DNA. Polymerase Chain Reaction (PCR) is typically used in order to create multiple copies of the desired DNA.

Ligation

Next, the isolated and amplified DNA will be placed into a vector in order to create recombinant DNA. In order to do this, ligation is needed. Ligation is the process of joining two different DNA strands together.

The vector needs to be opened using restriction enzymes in order to accept the DNA of interest. The DNA of interest also uses the same restriction enzymes in order to ensure their ends will be complementary to the opened vector. DNA ligase, another enzyme, will be used to attach the DNA of interest to the vector, which will create the desired recombinant DNA molecule.

Transformation

The recombinant DNA molecule is then given to a host cell, typically, bacterial cells are used, especially E. coli. A long procedure is done to ensure that the host cells take up the given recombinant DNA and are able to grow colonies containing this desired DNA. In some cases, these colonies will have to be kept all night in an incubator!

In order to make sure only the desired colonies grow, antibiotic resistance genes are typically used. For example, the recombinant DNA molecule could have an antibiotic resistance gene for ampicillin, and then the host cells are placed onto a petri dish containing agar gel with ampicillin. Only the host cells that contain the ampicillin resistance gene will be able to grow on the petri dish, which allows scientists to ensure that their transformation process actually worked.

Screening procedure

Lastly, the colonies from the petri dish will need to be harvested and actually checked in order to ensure that they have the desired insert. Typically, PCR will be performed again, or restriction fragment analysis will be used. Restriction fragment analysis uses restriction enzymes are used to digest isolated vector DNA. If the isolated vector DNA contains the desired insert, the restriction enzymes will excise it.

After PCR or restriction fragment analysis is used, the results will need to be checked using gel electrophoresis or DNA sequence analysis.

Gel electrophoresis is the process of using a gel, typically agarose-based, and electricity to separate DNA based on size and charge. DNA is inserted into small wells at the top of the gel. Negative charges, the black wire, occur at the top of the gel, and positive charges, the red wire, occur at the bottom. Since DNA is negatively charged, the negative charges at the top push the DNA down towards the positively charged gel bottom. The smaller the size of the DNA fragment, the farther it can travel down the gel. Desired DNA is typically assessed using a DNA ladder, bands of DNA with known values. DNA bands can either be measured in nucleotides (nt) or base pairs (bp), which are equal to each another. You can see an example of gel electrophoresis below in Fig. 3 with the DNA ladder on the left with its known values and three samples.

Figure 3. Example of gel electrophoresis. Source: Mckenzielower via wizeprep.com

DNA sequence analysis is a method that is able to assess the nucleotide structure of recombinant DNA. There are different types of DNA sequence analysis, and the type chosen depends on the size of the recombinant DNA. There are two main types of DNA sequence analysis:

• Sanger sequencing: Works for DNA up to 900 bp, but it is expensive and inefficient for anything larger.

• Next-generation sequencing: Works for DNA 50-700 bp, but it is faster, cheaper, and can run multiple sequences at once, unlike Sanger sequencing.

What are DNA cloning and assembly methods?

There are multiple different ways to clone DNA:

• Restriction enzyme based cloning: Uses restriction enzymes to cut the DNA fragment of interest and the vector and then use DNA ligase to place the DNA fragment into the vector.

• PCR cloning: Uses DNA ligase to place amplified DNA fragments of interest into the vector. A specific "T-tailed" vector is needed for this process which means the vector's ends must both be the nucleotide thymine.

• Ligation independent cloning: Matching sequences will be found on both the DNA of interest and the vector, and enzymes will be used to create the ends for the inserted DNA of interest and vector, so the annealing step will allow the ends to close and create the recombinant DNA molecule.

• Seamless cloning: Similar to ligation independent cloning, it needs an enzyme to create the ends of the DNA of interest and vector, but it needs DNA polymerase to fill in the gaps of the inserted DNA of interest and vector and DNA ligase to ensure the recombinant DNA molecule has been properly created.

• Recombinational cloning: A multistep process that uses an entry vector and DNA recombinase enzymes. The DNA of interest is placed into the entry vector. The entry vector is then combined with the destination vector to form what is called a destination clone using the DNA recombinase.

DNA cloning techniques

There are different tips and tricks to ensure DNA cloning efficiency. For example, it is key to making sure that DNA does not have contaminants that can reduce final cloning results. DNA contaminants can be removed by using purification kits. When performing restriction enzyme based reactions, it is important to know not to add too many restriction enzymes. The volume of restriction enzymes should not exceed 10% when compared to the total volume. Also, use quantitative measurement methods to assess whether or not there is the proper amount of material that will be used for downstream reactions.