In recombinant DNA technology, multiple DNA molecules are combined together and then introduced into a host cell to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. The use of restriction enzymes or restriction endonucleases is essential in the process of recombinant DNA technology. A restriction enzyme is an endonuclease that recognises specific base sequences called recognition sites in the DNA molecule and cuts at those sites to produce restriction fragments.
Depending on the size of the recognition sequence and where the enzyme cuts, a single restriction enzyme may produce sticky ends or blunt ends when cutting a piece of DNA. Enzymes are proteins that catalyze reactions. Enzymes can be derived from animals, plants, bacteria, and viruses.
The first step in recombinant DNA technology is cutting plasmids or bacteriophages by one or several restriction enzymes to generate cohesive and sticky ends. A gene is inserted into these sites by ligation with T4 ligase before transformation into competent cells for expression of recombinant protein or genomic library construction. Boster offers robust experiments on DNA technology using these enzymes discussed below.
Enzymes Used in Recombinant DNA Technology
There are several enzymes used in recombinant DNA technology, they include:
- Restriction enzymes
- DNA polymerases
- DNA Ligase
- Reverse Transcriptase
Restriction enzymes (also called restriction endonucleases) are a class of proteins that cut DNA at specific sequences. They recognize a specific DNA sequence and then cleave the DNA at that sequence. The enzyme is produced by bacteria, but once released into the cell it can break down any kind of foreign DNA that may be present in that cell.
There are two types of cuts that can be made by restriction enzymes:
In site-specific cleavage, a single strand of nucleotides is cleaved while leaving intact its complementary counterpart on the other end of the duplex DNA molecule; this results in linear fragments which contain single strands of nucleotides at both ends (the cohesive ends).
Non-site-specific cleavage occurs when both strands at one end remain intact after being severed from each other but both strands at another end have been removed by a restriction enzyme; this produces double stranded fragments with free 3′ 5′ overhangs(unpaired nucleotides).
Proteases are enzymes that break down proteins. Proteases are commonly used in custom recombinant protein production because they are able to digest the protein backbone, leaving only the functional domains (domain proteases). In addition to helping create new protein products, proteases serve other functions in the lab: they can be used to cleave specific sites within a protein (site-directed protease) or digest large amounts of unwanted material (mass spectrometry detergent).
These enzymes catalyze the synthesis of DNA. These enzymes catalyze the formation of new complementary strands of deoxyribonucleic acid (DNA) from existing templates. DNA polymerase as an enzyme helps with DNA replication. DNA polymerases are proteins that make up the core of the cell’s machinery for replicating DNA and repairing damaged DNA. There are many types of DNA polymerases, each with different functions in the cell: some copy genes from one place on a chromosome to another, others proofread newly copied strands or repair them when mistakes happen, still others do error-free repair when errors occur during replication or replication stress damages the double helix. The two main types of polymerase enzymes include:
The chain-elongating polymerases synthesize new products from existing templates until they encounter terminator nucleotides , which signal them to stop adding new nucleotides onto their growing strands.
Branch-terminating polymerases add one or more nucleotides at specific sites along existing oligonucleotide chains by recognizing specific signals within their templates.
DNA ligase is an enzyme that joins two pieces of DNA together. It’s used in many different scientific fields, including recombinant DNA technology to join DNA molecules that have been cut with restriction enzymes, cloning, and replication. DNA ligase also has applications in molecular biology, genetics, and other areas of study.
DNA ligases are found in both prokaryotes (bacteria) and eukaryotes (animals). The most common form is called T4 DNA Ligase; this type is used to help repair cuts or breaks in double-stranded DNA (dsDNA) molecules as part of the natural process of replication. The T4 DNA Ligase was isolated from E. coli bacteria by Arthur Kornberg at Stanford University in 1967 during his Nobel Prize winning research on how cells replicate their own genetic material.
For example, if you wanted to insert a gene for beta-galactosidase into a plasmid, you could use ligase to join your desired gene and the plasmid. You can also use ligases for the joining of two pieces of DNA(cloning). This happens when a cloned piece of DNA is amplified using PCR. It will make more copies of itself than one without ligase because there’s more than one molecule present.
Reverse transcriptase is an enzyme that catalyzes the formation of DNA from RNA. It is used in recombinant DNA technology, a process that uses pieces of DNA from different organisms and combines them to form new genetic sequences. Reverse transcriptase is used in the conversion of messenger RNA (mRNA) to complementary DNA (cDNA), and it can be used for other applications such as sequencing, microarray analysis, and gene expression profiling.
Reverse transcriptase was originally discovered by Dr. David Baltimore as a method for converting viral RNAs into double stranded DNAs so that their structure could be determined. Reverse transcriptase uses a DNA template to copy an mRNA molecule back into dsDNA format. This reaction occurs in two steps:
- Conversion of the RNA template strand into a single-stranded DNA by incorporation of dNTPs 2. Addition of deoxynucleotides (dNTPs) using reverse transcriptase activity
The enzymes listed above play a crucial role in recombinant DNA technology and have made it possible for researchers all around the world to study genes, cure diseases, and manipulate crops.