Scientists use various molecular biology techniques to analyze DNA. In this article, I have summarized a few of them for you so you can better understand the general principles and steps involved in their operation.
Restriction Enzymes and Molecular Cloning
Many copies of a gene of interest can be made by isolating the gene, placing it in bacteria, and then growing the bacteria. As the bacteria grows, it will also replicate the gene of interest. The gene of interest or its protein product can then be isolated. A restriction enzyme is used to cut the gene of interest from DNA. After that, a bacterial plasmid containing a biomarker is cut with the same restriction enzyme. The cut in both the parent DNA and plasmid have overhangs or “sticky ends” that complement each other. In the next step, they are ligated together to form a recombinant plasmid. This plasmid is then placed in bacteria by a process called transformation. As the bacteria grow, the gene of interest is replicated and can later be isolated.
Molecular Cloning Using PCR
In cases where a small amount of DNA is available, using PCR for cloning is a better approach. This method does not need restriction enzymes. The process involves the rapid amplification of the gene of interest in the first steps, followed by ligation into a vector (plasmid) as above. The vector can then be placed in bacteria and then grown to replicate the gene of interest.
A DNA library is a collection of DNA fragments that represent the entire genome of an organism. The DNA is stored in a population of identical vectors, each with a fragment of the DNA. It is constructed by first extracting the DNA and then digesting it with restriction enzymes to make many fragments. The fragments are then inserted into a vector (plasmid) and then transferred to a host such as E. coli for amplification. Using this library, scientists can isolate and study specific colonies that have the genes they are interested in studying.
Southern blotting is a technique used to detect a specific sequence of DNA in a complex mixture. The process starts with cutting the DNA into fragments using restriction enzymes and then separating the fragments by size using gel electrophoresis. A mild alkali (NaOH) is then used to denature the DNA, transforming it from double-stranded to single-stranded. The DNA is then transferred to a nylon or nitrocellulose membrane, after which a probe is added. Since the probes are complementary to the gene of interest, only those genes will be hybridized. The hybridized genes can then be visualized using autoradiography, fluorescence, or color change, depending on the type of probe used.
Northern blotting determines the presence of RNA, which ultimately tells whether a gene is expressed. The first step is extracting the RNA and denaturing it with formaldehyde to remove base pairing (remember that RNA, although single-stranded, can base pair with itself). The RNA is then separated by gel electrophoresis, transferred to a membrane, and then treated with a radioactive probe. The probe binds to the RNA of interest and can be visualized using an autoradiographic method.
Sanger sequence is a method that is used to determine the letter sequence in DNA. During the process, both deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) are incorporated by DNA polymerase into the DNA to be copied. Since ddNTPs lack a hydroxyl group at the 3’ end, termination occurs when they are added. This creates fractions of different sizes, with the last nucleotide on each fragment representing a ddNTP. The fractions can then be separated using electrophoresis. Since the last letter of each fragment is known, the correct sequence can be determined by reading the gel from bottom to top (smallest fragment to largest). Here are the key steps in the process –
- Amplify the DNA using PCR
- Add amplified DNA in a reaction vessel containing
- DNA polymerase
- The four normal nucleotides (A, T, G, and C)
- Small concentrations of the four probe-linked modified nucleotides (ddATP, ddTTP, ddGTP, and ddCTP)
- Allow DNA polymerase to copy DNA in the reaction vessel
- Separate the resulting fragments by electrophoresis
- Write down the sequence by reading the gel from bottom to top