Chapters
In this article, we will describe and explain how gel electrophoresis is employed to separate DNA fragments of different lengths. Furthermore, we will also outline how microarrays are used in the analysis of the genome and the detection of mRNA in studies of gene expression. So, let us get started.
Gel Electrophoresis
Gel electrophoresis refers to a method that is commonly employed in analyzing DNA, RNA, and proteins. During this process, the molecules get separated as per their size or mass and net (total) charge.
Why separation takes place?
The separation takes place because of the following reasons:
- The molecules carry an electrical charge. Out of these molecules, the positively charged molecules move towards the cathode, i.e. the negative pole and the negatively charged molecules move towards the anode, i.e. positive pole. For instance, since DNA is a negatively charged molecule because of the presence of phosphate groups, hence when it is put in the electrical field, its molecules will move towards the anode.
- The molecules have different sizes that move through the gel at varying rates. The small pores in the gel lead to quick movement of the smaller molecules. On the other hand, the larger molecules move slowly.
- The pore sizes of different gels vary which affects the speed at which the molecules can move through them.
DNA Separation
DNA samples can be taken from almost anywhere in the body. For instance, we can get them from the root of the hair or saliva from the cup. After collecting the DNA, it should be prepared for gel electrophoresis to sequence or analyze it for genetic profiling (fingerprinting).
How fragments are prepared?
In order to prepare the fragments, scientists should first enhance the number of DNA molecules through the polymerase chain reaction (PCR). Then they employ restriction endonucleases to cut DNA into fragments.
What is VNTR (variable number tandem repeat regions)?
Different restriction enzymes cut the DNA at varying base sequences. Hence, scientists use enzymes that will cut near the VNTR. The VNTRs are the regions present in the non-coding part of DNA. They have different numbers of repeated DNA sequences and are different in different people except for identical twins. These VNTRs can be called satellite or microsatellite DNA.
How are DNA fragments separated in gel electrophoresis?
Scientists follow the procedure listed below to separate the DNA fragments in gel electrophoresis:
- In a tank, create an agarose gel plate and cut the wells (a series of groves) into the get at one end
- In an electrolyte solution, submerge the gel in a tank. An electrolyte solution refers to a salt solution that conducts electricity.
- By employing a micropipette, insert or load the fragments into the wells
- Apply electrical current to the tank in such a way that the negative electrode is connected to the end of the plate with the wells. This is because the DNA fragments will move towards the anode, i.e., the positive pole due to the attraction between the phosphates which are negatively charged, and the anode.
- The smaller mass or shorter pieces of DNA fragments will move quickly and away from the wells as compared to the larger fragments.
- The fragments cannot be seen, hence they should be moved onto absorbent paper of nitrocellulose. This paper then undergoes heating to separate the two DNA strands. After that, probes are added followed by taking an X-ray image or shining UV light onto the paper to produce bands which are usually compared to the control fragment of DNA.
What are Probes?
Probes refer to the single-stranded DNA sequences that are complementary to the VNTR regions needed by scientists. They also contain a method by which they can be identified. This method can be either a radioactive label or a fluorescent stain.
- A radioactive label: For instance, a phosphorus isotope that forces the probes to emit radiation that makes the X-ray film go dark by creating a pattern of dark bands.
- A fluorescent stain or dye: For instance, ethidium bromide which glows when exposed to UV light and creates a pattern of colored bands.
Protein Separation
The charge of proteins is determined by different amino acids. The charge of the R groups is determined by the pH and hence to keep the pH constant, buffer solutions are utilized during the separation process. Gel electrophoresis is employed to separate the polypeptide chains produced by different alleles. For instance, the hemoglobin variants like α-globin, β-globin, and the sickle cell anemia variant of β-globin.
Microarrays
Microarrays refer to the laboratory tools that are used to identify the expression of thousands of genes at the same time. They are also used to detect the presence of genes in the genome of an organism.
Uses of Microarrays
- They are used in the diagnosis and treatment of medical conditions, biotechnology, and forensic analysis.
- Microarrays are extremely useful for scientists because they allow them to study a large number of genes in a short period.
Probes in Microarrays
A microarray contains a tiny usually 2cm2 piece of plastic, glass, or silicon (also referred to as chips) to which probes are attached to a spot known as a gene spot in a grid pattern. Probes refer to the short lengths of single-stranded DNA or RNA which undergo synthesis so that they can be complementary to a particular base sequence.
How are Microarrays used to analyze genomes?
This is how microarrays are used to analyze genomes:
- DNA samples taken from the species need to be compared
- Using the restriction enzymes, DNA is cut into fragments
- The DNA fragments are then denatured to create single-stranded DNA molecules
- Using the fluorescent tags, the DNA fragments are then labeled
- After mixing these DNA fragments, they are allowed to hybridize with the probes on the microarray
- After some time, any DNA left that did not undergo hybridization with the probes is washed off
- The microarray is then observed using an ultraviolet light
- The color indicates the place where hybridization has taken place because the DNA fragment is complementary to the probe. If red and green, fluorescent spots are observed, then it means that only a single species of DNA has hybridized. On the other hand, the yellow spot color indicates that both species have hybridized with that DNA fragment. It implies that a gene is present in both the species
- If the spot does not have any color, then it shows that the gene is not present in either species.
Transcription and Translation
When the genes are in their active form or are expressed, then several copies of mRNA are produced by transcription. After that, the corresponding proteins are produced from these mRNAs during translation. Hence, scientists can indirectly determine the type of genes being expressed in the cells by evaluating the number of mRNAs.
How are microarrays used to detect whether a gene is being expressed?
Microarrays identify the quantity of mRNA present to detect whether a gene is being expressed or not. The following steps are taken to compare which genes are expressed using microarrays:
- Collect mRNA from both types of cells and use the reverse transcriptase to convert mRNA to cDNA
- You may use the PCR to enhance the quantity of cDNA
- Add fluorescent tags to the cDNA
- After that, denature the cDNA to produce single-stranded DNA
- Allow the single-stranded DNA molecules to hybridize with the probes on the microarray
- Shine the UV light on the microarray. The spots that will shine will reflect that the gene was transcribed, i.e. expressed and the intensity of the light coming from the spots will show the quantity of mRNA being produced.
- If the emitted light has high intensity, then it indicates the presence of many mRNAs. On the other hand, a low-intensity light indicates that only a few mRNAs are present
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