There are several experimental techniques to detect and quantify nucleic acids in your sample. These include PCR, RT PCR, sequencing and blotting. There are also techniques such as EMSA, footprinting, ChIP and probing that detect inter-molecular interactions of nucleic acids.
Research in nucleic acids involves the isolation and characterization of DNA and RNA molecules from various cells, tissues, and organisms.
Nucleic acid research aims to figure out the physiological roles played by these macromolecules; for example, how they participate in replication, transcription, and translation. This research can provide insights into the genetic basis of traits and diseases, cell signaling and behavior, as well as the mechanisms underlying gene expression and regulation.
In this article, we will take a quick look into two aspects of nucleic acid research, focusing on the experimental approaches used in each of these domains:
A) detection and quantification of nucleic acids in your sample
B) detection of nucleic acid interactions with other biomolecules
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Detection and quantification of nucleic acids is an important aspect of all areas of modern bioscience research, including molecular biology, genetics, and microbiology.
Nucleic acids like DNA and RNA have genetic information and are involved in many biological processes. There are various techniques used for the detection and quantification of nucleic acids, depending on the questions being asked.
PCR stands for Polymerase Chain Reaction. It is a very commonly used laboratory technique that allows amplification of a specific DNA segment.
Kary Mullis first developed the technique in the 1980s, and this technique has indeed revolutionized molecular biology and genetics.
PCR is a powerful technique because it produces millions of copies of a specific DNA sequence from a just a tiny amount of amount of starting material, such as DNA from a single cell or a tiny tissue sample.
Even a single molecule of your required DNA fragment is enough to be used as the template for a PCR.
PCR is used in so many different research applications including biotechnology, medicine, diagnostics and forensics. It can be used to identify genetic mutations, diagnose diseases, detect infectious agents, and analyze DNA from crime scenes or ancient specimens.
RT-PCR (Reverse Transcription Polymerase Chain Reaction) is pretty similar to PCR in that both methods are used to amplify nucleic acid molecules. The difference between PCR and RT-PCR lies in the starting molecule that serves as the template for amplification.
As you know, transcription is the process by which genetic information from a strand of DNA is transcribed into mRNA. Reverse transcription, as the name suggests, is just the opposite. Here, you start with an RNA molecule.
In PCR, the starting template is DNA whereas in RT-PCR, the starting template is an RNA molecule.
Using this single-stranded RNA molecule as the template, double-stranded complementary DNA (cDNA) molecules are synthesized. The synthesis of cDNA molecules which is catalyzed by reverse transcriptase, an RNA-dependent DNA polymerase, is an additional step. This is followed by the usual PCR reaction that amplifies the newly synthesized DNA.
RT-PCR is a commonly used laboratory-based technique that helps researchers study gene expression. It helps detect RNA transcripts in a cell or tissue and the concentration of a specific type of RNA molecule that is being assayed for.
Quantitative RT-PCR is another variant of RT-PCR. Researchers use this technique to quantify the target gene. In molecular medicine, where real quantification of RNA targets is critical, qRT-PCR is the go-to technique.
RT-PCR and qRT-PCR have a multitude of applications, including diagnosis of viral infections, diagnosis of genetic diseases, and detection of genetic mutations in cancer research. RT-PCR has become especially important during the COVID-19 pandemic because it is used to detect SARS-CoV-2 RNA in patient samples to confirm infection.
Also, the amount of viral load in patients affected by retroviruses can be ascertained by measuring viral RNA levels using qRT-PCR.
Sequencing refers to detecting the specific order of nucleotides (A, G, C, T) in a target DNA or RNA molecule. The DNA sequencing process involves analyzing DNA fragments to determine the nucleotide sequence, while RNA sequencing involves determining the sequence of RNA molecules produced in a cell.
The ability to sequence DNA has revolutionized molecular biology by providing a powerful tool for studying the structure, function, and evolution of genes, as well as diagnosing and treating diseases.
Sequencing enables discovery of new genes and variants to provide insights into evolutionary relationships between species.
Here are some of the areas where sequencing has transformed molecular biology.
- Studying genetic diseases: In order to identify any genetic mutation or variation that might be responsible for causing a specific disease in a patient, scientists heavily rely on gene sequencing techniques.
- Drug development: Sequencing helps predict drug targets within a genome. Once the target sequence is determined, it helps predict how a drug will interact with a patient’s DNA.
- Evolutionary studies:Scientists use sequencing to determine the percent homology and similarity in the sequence between different species. This helps to determine how different species are related to each other and how they have evolved over time.
- Modern personalized medicine: Sequencing can help doctors determine the best treatment plan for a patient based on their unique genetic makeup.
These are just few examples from the vast horizon where sequencing is so very helpful from the fundamental steps of molecular biology such as cloning into a plasmid to translational such as figuring out if a patient’s tumor has a certain mutation.
There are various methods of DNA and RNA sequencing that differ in their cost, throughput, and accuracy. Sanger sequencing, next-generation sequencing, and single-molecule sequencing are some of the techniques that are most commonly used.
Spectroscopy is used to quantify the concentration of macromolecules such as nucleic acids in your sample.
It involves studying the interaction between light and matter where light is separated into its component wavelengths and the resulting spectrum is then used to characterize various properties of the sample being studied.
Some of those properties include the chemical composition, physical properties like pressure and temperature, and molecular structure of the sample. The properties of light that are emitted, absorbed, or scattered by the matter are measured using a spectrometer.
Spectroscopy is used to study an extensive range of physical, chemical, and biological phenomena.
A spectrophotometer is another tool in spectroscopy that can be used to measure the concentration and purity of DNA and RNA samples at specific wavelengths at which these nucleic acid molecules absorb UV light.
The absorption spectrum of DNA and RNA molecules is characterized at two major peaks: one at 260 nm and the other at 280 nm. The ratio of the absorbance at 260 nm to that at 280 nm helps estimate how pure the DNA and RNA sample is.
For your DNA sample, the closer the A260/ A280 value is to 1.8, the better it is for downstream experimental procedures.
Any value greater than 1.8 indicates possible RNA contamination in your DNA sample because A260/ A280 =2.0 for pure RNA samples.
Any value less than 1.8 indicates the presence of other impurities like proteins, insoluble cell lysate factors, etc. within the DNA sample.
Southern blot, a technique named for its inventor Edwin Southern in 1975, is a molecular technique used to detect target DNA sequences within a sample.
The process of Southern blotting involves extraction of your DNA sample of interest, digestion with restriction enzymes, and gel electrophoresis. This is followed by transfer of DNA fragments from the gel to a membrane and hybridization with a complementary DNA probe.
The target sequence hybridized to the probe is then detected by visualizing the probe on the membrane using autoradiography, fluorescence, or chemiluminescence.
Southern blotting is a versatile technique that can be used in different applications in molecular biology and genetics including:
- detection of genetic mutations associated with various diseases for example, sickle cell anemia.
- creation of DNA fingerprints, which are unique patterns of DNA fragments that can be used to identify individuals. This is a great tool used in forensic science and paternity testing.
- confirming the integration of a transgene within the genome of an organism.Analyzing the expression and function of a transgene.
- detect viral DNA in infected cells which helps diagnose viral infections and in studying viral replication.
Northern blot, developed in 1977, is a molecular biology technique that helps detect levels of RNA molecules of a specific sequence in your sample. Northern blot helps monitor target gene expression in a cell or tissue sample, by assaying for the corresponding mRNA transcript.
The principle of Northern blot is similar to Southern blot, only the macromolecule being studied is different.
The first step in a Northern blot involves the separation of RNA molecules based on their molecular size by gel electrophoresis. This is followed by blotting or transferring the separated RNA samples onto a membrane, and then detecting the specific target RNA molecule by using a complementary DNA or RNA as a probe.
Northern blotting allows researchers to study the expression of specific genes under different physiological conditions, even in diseased or abnormal conditions. It also helps analyze the abundance of target RNA molecules during different developmental stages in cells, tissue, or organ.
In cancer research, this technique helps detect oncogenes that are overexpressed in cancer tissue compared to normal tissue.
Northern blot may sometimes be substituted by other techniques like RT-PCR, especially in labs that avoid using radioactivity.
Nucleic acids interact with various biomolecules such as proteins, lipids, carbohydrates, and other nucleic acids. Studying these interactions helps us discover the biological functions of nucleic acids and helps us develop new therapeutic and diagnostic approaches.
Some of the most commonly used methods to study the interaction of nucleic acids with other biomolecules are EMSA, ChIP, probing and DNA footprinting.
Electrophoretic mobility shift assay (EMSA) or gel shift assay is a molecular biology technique used to study the interactions between nucleic acids and proteins, or to study nucleic acid-nucleic acid interactions.
It helps detect whether a particular protein or nucleic acid of interest binds to the nucleic acid (DNA/RNA) being studied at a given time based on the electrophoretic separation on an agarose or polyacrylamide gel.
With EMSA, the nucleic acid under study is labeled with a radioactive or fluorescent or biotin tag and mixed with the protein or nucleic acid of interest.
During gel electrophoresis, the nucleic acid-protein complex will migrate more slowly than the unbound nucleic acid fragment resulting in a shift in the mobility of the DNA bands. In some instances, there might be more than two protein molecules bound to the nucleic acid at a given point of time.
This shift can be visualized using fluorescent imaging or autoradiography. The intensity of the shifted band can be used to determine the strength of the protein-nucleic acid interaction.
Chromatin immunoprecipitation (ChIP) is a technique to study protein-nucleic acid interactions.
It involves cross-linking of protein-DNA complexes in living cells. This is followed by immunoprecipitation of the protein of interest using specific antibodies.
The DNA associated with the protein is then isolated, purified, and sequenced for downstream analysis.
ChIP provides valuable insights into the regulation of gene expression and the role of specific proteins in cellular processes. It is a useful tool in epigenetic studies where it helps detect the exact location of a target DNA sequence bound to the chromatin and other histone modifications in a genome.
In molecular biology, probing is a technique that utilizes labeled probes to detect specific proteins of interest or protein-protein interactions, or protein-nucleic acid interactions.
DNA or RNA probes are single-stranded fragments of nucleic acid that are labeled with a detectable signal such as a radioactive isotope, a fluorescent dye, or an enzyme. These probes are designed to hybridize or bind specifically to the complementary sequences of DNA or RNA in a sample.
DNA footprinting is used to study the interaction between a DNA molecule and a protein of interest or other biomolecules.
To test this, you incubate your sample DNA with the protein of interest and a DNA-cleaving chemical like DnaseI.
DnaseI will cleave your DNA fragment.
In case the protein you are testing indeed binds to your target DNA fragment, then that segment of DNA where this protein binds would not be accessible to DnaseI; and as a result, it would not be cleaved.
In other words, the protein, by binding to this DNA segment, would protect it from getting cleaved. Theses binding sites appear as gaps or “footprints” in the pattern of cleaved DNA.
The resulting pattern of the cleaved and uncleaved regions of DNA can be visualized by gel electrophoresis. This reveals the specific binding sites of the protein of interest.
This technique is often used in molecular biology and genetic research to identify and study DNA-binding proteins such as transcription factors, and their interactions with your DNA fragment of interest.
It can also be used to study the structure and function of chromatin, the complex of DNA and proteins that make up the structure of the chromosome.
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