This article begins a new series about gene technologies. Many gene technologies, especially recombinant DNA technologies, begin with the need to isolate or make DNA fragments. We will look at three different ways that this can be achieved.
This method allows DNA fragments to be “cut out” of existing DNA. Restriction enzymes, sometimes called restriction endonucleases, are used to cut at specific recognition sequences. These sequences are palindromic regions of DNA (regions where the base pairs are antiparallel, so read the same in both directions). However, the restriction enzymes do not normally cut straight between base pairs. Instead, they usually cut to leave “sticky ends“, which are then free to complementary base pair with other DNA further on in processing such as cloning. There are lots of different restriction enzymes which are all specific to different recognition sequences, so you can decide which enzyme to use based on where you want to cut. Don’t forget that cutting DNA involves breaking phosphodiester bonds using a hydrolysis reaction.
This method enables DNA fragments to be produced based on an mRNA template. In every cell there are only two copies of each gene within DNA. This makes it difficult to isolate one gene. However, there are many copies of mRNA in a cell which are complementary to the gene (if it is being transcribed). So it is possible to isolate the mRNA from cells, and carry out reverse transcription to produce a DNA fragment from the mRNA template. The enzyme reverse transcriptase is capable of catalysing this process by joining free DNA nucleotides together. The DNA produced is called complementary DNA (cDNA). There are two differences between DNA found in the nucleus (genomic DNA) and cDNA:
- cDNA only contains exons, no introns. This is because the mRNA template does not contain introns as they are removed during splicing.
- cDNA is single stranded, not double stranded.
I’m sure whoever came up with the name “gene machine” is very proud of themselves. This technology enables you to produce DNA fragments from scratch and from your own design. You can make any sequence you like, you just have to tell the machine what base sequence you want. The gene machine is only able to make small sections of DNA, then it joins them together to produce the full DNA fragment.
DNA fragments can be produced by:
- Isolating a fragment from existing DNA using restriction enzymes.
- Creating a fragment based on an mRNA template using reverse transcriptase.
- Creating a fragment from scratch using a gene machine.