In the last 20 years, recombinant DNA technology has progressed greatly. In 2003, scientists sequenced all 3 billion bases of the human genome for the first time, costing around 3 billion dollars. Now we can sequence someone’s entire genome for about 100 dollars.
Synthesising DNA fragments is a vital tool in recombinant DNA technology. There are three methods to make DNA fragments. These are:
1) Using reverse transcriptase. Instead of taking the DNA fragment directly from DNA in the nucleus, which might be difficult. It can be easier to isolate the mRNA from the cytoplasm and turn it into complementary DNA (cDNA). This is achieved by mixing the isolated mRNA with free-floating DNA nucleotides as well as the enzyme reverse transcriptase, where the enzyme uses the mRNA as a template to synthesise new strands of complementary DNA.
2) Using restriction endonuclease enzymes. Some sections of DNA have palindromic sequences of nucleotides, where the base sequence can be read in the same order in the opposite direction. Restriction endonucleases are enzymes that recognise specific palindromic sequences (called recognition sequences) and cut (digest) the DNA at these sites. The specific recognition sequence of the restriction endonuclease depends on the enzyme’s active site.
If there are recognition sequences either side of the target DNA fragment, one or two types of restriction endonucleases can be used to separate it from the rest of the DNA. Some restriction endonucleases make sticky ends when they cut, which are small tales of unpaired bases at each end of the fragment, that can be used to bind a fragment with another piece of DNA.
3) Using a gene machine. More recently, technology has allowed for DNA sequences to be synthesised from scratch, so any sequence can be made. However, they are expensive.