Question

Hi, what are 3 benefits to using recombinant DNA over PCR as someone in the lab? So, I don't mean its applications per say (i.e. DNA Fingerprinting). I want to know what is it about each that make what I would choose for a particular job (i.e. in addition to PCR ideal for discrete regions vs gene libraries and expression vectors for recombinant DNA). Cost and ease of processes is already understood).

Answer 1

Recombinant DNA and PCR, both have variegated functionality that can be used in different setups, either together or separate. In addition to what you have mentioned,
For in case of Recombinant DNA:

• If we want a particular protein to assess, be it it's structure/activity, we need to first extract it. One of the ways to do it, is create a recombinant vector using it's cDNA ligated to it, and expressed in bacterial systems, most commonly BL21 strain, where the mRNA stability is enhanced. To overexpress a prokaryotic gene in large numbers, manipulation can be done by using a strong promoter which is inducible.
• Gene therapy is massively conducted using rDNA. Normal genes are introduced into defective cells to ensure a normal protein product. It has been past conducted for defects like SCID, where retroviral vector(containing ADA) infection in patient's stem cell has been conducted, which has been transferred to patients again. So, if you are studying a defect in a particular organism, and it is suspected to be for the absence of a certain protein, out a bigger intracellular network. It can be done by using a knock-out mouse as a model.
• If you are working on a certain infection, and a vaccine needs to be synthesized, rDNA technology is used massively to make vaccines out of potent surface molecules.
• In order to make a herbicide/drought/flood resistant plant, rDNA technology can be used to increase the competence.

Other than the things you mentioned, PCR can be also used for:

• In order to see the difference between a particular protein expression in normal and diseased cells, quantitative PCR can be used and confirmed for it's over or under-expression that can help to deduce a directed conclusion.
• In order to understand a particular mutation, allele specific PCR can be used, where the inner forward primer can have a base change conforming to the mutation in the particular gene in the 3'end. This way, we can even differentiate between diseased and normal cell states, looking at the length of the gel bands.
• You can even introduce mutations and asses the importance of a particular sequence in a protein (either as a folding propellant/location signalling sequence etc) by site directed mutagenesis using PCR. Additional information is, use high fidelity polymerases to avoid secondary mutations.