What do you think is important in the amplification of gene of interest?

Question

Edit: I know it is about PCR, but I do not know what should I look out for when doing PCR so that I can amplify my gene of interest successfully.

Answer 1

it's Polymerase chain reaction (PCR)
PCR is a molecular biology technique [1] for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA and it can be extensively modified to perform a wide array of genetic manipulations.

PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 1 one night in his car. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in Chemistry with Michael Smith in 1993.

As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."

Answer 2

In amplifying the gene of interest there are two main ways. For small genes the whole thing can be amplified by PCR (as described by the above answer) in which case the position and composition of the primers is of utmost importance along with the conditions of the reaction itself to ensure there is good amplification and no unwanted products. For larger genes many PCR reactions can be carried out to obtain the full sequence or alternatively the gene could be cloned in to a vector (usually a plasmid) and inserted in to a bacterium. The bacteria can then be grown up and the plasmid extracted and purified. There are many factors to consider when using a cloning method to produce optimal yields.

Answer 3

The most important thing is to use the right primers and where possible, use good quality specimens.

If you're using PCR then the gene of interest has to be fairly small. If its really long, then gene cloning will be a better option.

If there are no primers available for the gene of interest then the only option is gene cloning.

Answer 4

to find the DNA sequence is doing Gene Amplification.