Isolating and Amplifying Specific DNA fragments

How can a specific segment of DNA be isolated from an entire genome?Furthermore, how can it be isolated in quantities sufficient to analyze features of the DNA such as its DNA sequence and its protein product? An important approach was that researchers could make the large samples of DNA that they needed to isolate a gene by tricking the DNA replication to replicate the gene of interest. Such replication is called "amplification". It can be carried out in vivo (in live bacterial cells)  or in vitro (in a test tube).

In the in vivo approach, an molecular biologist starts with a sample of DNA  containing the gene of interest. This sample is called "donor DNA", and mostly it is a very large (or entire DNA genome) DNA molecule.Fragments of the donor DNA are inserted into a specially designed plasmid or bacterial virus that will “carry” and amplify the gene of interest and are hence called "vectors".

First of all, the donor DNA molecules are cut up by using enzymes called restriction  enzymes (restriction endonucleases also called - molecular “scissors.”) They cut long DNA molecules into hundreds of fragments of more manageable size.

Next step, the fragment is inserted into a cut vector chromosome to form recombinant DNA molecules. The recombinant DNA molecules are then transferred into bacterial cells, and, generally, only one recombinant molecule is taken up by each cell. The recombinant molecule is amplified along with the vector during the division of the bacterial cell. This process results in a clone of identical cells, each containing the recombinant DNA molecule, and so this technique of amplification is called DNA cloning. The next stage is to find the rare clone containing the DNA of interest.

In the in vitro approach, called the polymerase chain reaction (PCR), a specific gene of interest is isolated and amplified by DNA polymerase. PCR “finds” the target DNA fragment by the complementary binding of specific short primers to the ends of that sequence. These primers then guide the replication process, which cycles exponentially, resulting in the production of large quantities of the target DNA as an isolated DNA fragment. Even larger quantities of target DNA can be obtained by inserting the PCR product into a plasmid, thus generating a recombinant DNA molecule like that described above.

Two methods of isolating and amplifying a gene. (a) in vivo, by tricking the replication machinery of a bacterium into amplifying recombinant DNA containing the gene, and (b) in vitro, in the test tube using the polymerase-chain-reaction technique. Both methods employ the basic principles of molecular biology: the ability of specific proteins (yellow) to bind to DNA and the ability of complementary single-stranded nucleic acid segments to hybridize together (the primer used in the testtube method).