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| Year : 2014 | Volume
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| Issue : 1 | Page : 91-94 |
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| Procedures to view aberrations-A travel from protein to gene: Literature review |
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B Premalatha1, V Ramesh1, SPK Kennedy Babu2, PD Balamurali1
1 Department of Oral Pathology and Microbiology, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Indira Nagar, Pondicherry, India
2 Department of Periodontics, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Indira Nagar, Pondicherry, India
Click here for correspondence address and email
| Date of Submission | 15-Dec-2011 |
| Date of Decision | 18-Jan-2013 |
| Date of Acceptance | 12-May-2013 |
| Date of Web Publication | 21-Apr-2014 |
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 Abstract | | |
The diagnosis of any pathology is fundamentally based on the microscopic structure of cells and tissues and this remains as the standard by which all other diagnostic tests are measured. In this era, the pathologists are relying on the examination of tissue section stained by histochemical means and it is supported by the advanced immunological, biochemical and molecular techniques. This review will provide the information about one of the way that can be followed to unravel the molecular mechanism in spotting the disease process. Technologies used to study the cellular process are same for the normal and the abnormal cell. Experimental strategy briefed here is also applicable for both. The cellular process can be studied either from protein to gene or from gene to protein. Earlier days biochemical analysis (isolation of protein, protein sequencing) was separate and genetic analysis (genomic mapping) was separate. But now with advent of recombinant DNA technology it is possible to have a link between the biochemical and genetic analysis. Intermediary step of development of oligonucleotide synthesis, complementary DNA probe and cloning has revolutionized the research process. Identified gene can be compared with the normal gene by comparative genomics or expressed proteins by expression proteomics. Keywords: Gene, molecular aberration, protein
How to cite this article:
Premalatha B, Ramesh V, Babu SK, Balamurali P D. Procedures to view aberrations-A travel from protein to gene: Literature review. Indian J Dent Res 2014;25:91-4 |
How to cite this URL:
Premalatha B, Ramesh V, Babu SK, Balamurali P D. Procedures to view aberrations-A travel from protein to gene: Literature review. Indian J Dent Res [serial online] 2014 [cited 2023 Sep 23];25:91-4. Available from: https://www.ijdr.in/text.asp?2014/25/1/91/131145 |
The shapes and functions of cells are determined by their proteins. Proteins are generally synthesized on free ribosomes in the cytoplasm or on the rough endoplasmic reticulum. Among the proteins synthesized in the ribosomes, many proteins remain within the cell to carry out their functions and some proteins move to the membranes are responsible for the transport of molecules to different site. [1] Proteins in membranes are responsible for the transport of molecules from one cellular compartment to another and between inside and outside of the cell. Synthesis of protein itself is catalyzed by proteins, but this process is ultimately governed by deoxyribonucleic acid (DNA), which carries all information needed to specify the structure of every protein the cell can make. [2] The way in which cells store and express their genetic information has an enormous impact on many fields of study, such as pathology, immunology, medicine, microbiology and forensic science. DNA is a very big molecule, comprises approximately 3,000,000,000 bp (3000 mega base pair) in a haploid genome. Only about 1-2% of the human DNA contains genes; the rest are silent areas. About 1% of DNA is present inside mitochondria. There are only about 25,000-30,000 protein coding regions in the human DNA. Thus, most of the DNA is made up of non-coding sequences. [3]
The nuclear genome is divided into linear DNA molecules, the shortest 55 Mb in length and the longest 250 Mb, each contained in a different chromosome. DNA is a double stranded molecule in the shape of double helix. A crucial feature of this structure is that it depends on the sequence of bases in one strand being complementary to that in the other, in which thymine can form hydrogen bonds with adenine and cytosine with guanine in such a way that the distance between the 1' carbon atoms of their respective deoxyriboses are same. [4]
Once the structure of DNA and genetic code were understood, it became clear that many deep biological secrets were locked up in the sequences of bases of DNA. But identifying the sequences of long regions of DNA and modifying them at will, seemed a distant dream. How does one find and study a particular gene among the thousands of genes nested in the billions of base pairs of mammalion genome? Solution began to emerge in 1970s. [5] A series of technical discoveries in the 1970s drastically changed this perspective and has led to remarkable advancement in analysis and manipulation of macromolecules such as protein, DNA, and ribonucleic acid (RNA). [6]
The discovery of two types of enzymes (restriction enzymes and DNA ligases) provided the impetus for the recent development in recombinant DNA and permit DNA cloning. Any cloned DNA segment whether natural, modified or completely synthetic can be reinserted into the cells and can be tested for its biological activity. This group of techniques, often collectively referred to as recombinant DNA technology became the dominant approach for studying many basic biological procedures. In this review, it is planned to discuss the various procedures that can be utilized to identify the gene from the isolated functional protein of interest. [5],[6]
| Different Procedures to Identify a Gene From Protein | |  |
In the past, researchers had two basic approaches for unraveling the molecular mechanisms underlying various cellular processes. One approach involves the biochemical purification and analysis of a specified protein based on its functional characteristics by techniques such as electrophoresis and chromatography. [7] The other approach involves the characterization and mapping of genes defined by mutations using classical genetic analysis, like chromosomal mapping to locate the mutations on particular chromosomes, abnormalities of the chromosome mapped by banding analysis and restriction fragment length polymorphism. With the recombinant DNA technology it is possible to unravel from protein to gene or from gene to protein. The combination of biochemical and genetic approaches by recombinant DNA technology provides an enormously powerful strategy for studying the role of particular protein in complex processes. Design and development of chemicals and equipment facilitated the growth of this technology, which paved the way for analysis of biological molecules. This review has focused on the experimental strategy of starting with a protein identified and purified by biochemical techniques and eventually isolating the gene encoding it.
To understand procedure of isolating gene from protein, involves different steps the details of which are given in [Figure 1]. The following are different steps involved in identifying gene from the isolated protein:
- Isolating the protein
- Protein sequencing
- Designing the oligonucleotide probe
- Choosing the clone from genomic/complementary DNA (cDNA) library by membrane hybridization technique
- DNA sequencing.
 | Figure 1: Flow chart for the techniques to be used while identifying a gene from protein
Click here to view |
| Techniques for Isolating the Protein | | |
Isolation of protein (majority of enzymes are protein) is done by employing various simplified procedures such as centrifugation, electrophoresis, and liquid chromatography. [7],[8]
- Centrifugation
- Differential centrifugation
- Rate zonal centrifugation
- Equivalent gradient centrifugation.
- Electrophoresis
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE analysis)
- Two-dimensional gel electrophoresis.
- Liquid chromatography
- Gel filtration chromatography
- Ion-exchange chromatography
- Affinity chromatography
- High-performance liquid chromatography.
| Protein Sequencing | | |
Sequence of a protein is like a fingerprint, it uniquely establishes the identity of a protein (i.e. the linear order of aminoacid). Protein sequencing is determined by chemical method in which polypeptides are cleaved at its peptide bond. Edman degradation method, which is identified by high pressure liquid chromatography determines the amino acid sequence.
Identification of N-terminal amino acid sequence of the isolated protein of interest by Edman degradation involves a repetitive procedure [Figure 2]. In the first step of coupling the polypeptide is reacted with phenyl isothiocyanate which forms a covalent bond with the N-terminal amino group. In the second step, the coupled N-terminal amino acid is cleaved from the polypeptide by acid hydrolysis (acid cleavage), in which cyclic phenylthiohydantoin (PTH) derivative of the N-terminal amino acid (lysine represented by K) is separated and the polypeptide that is shorter at its N-terminus by one residue (NH 2 -S-L-V-E-E-COOH). These two steps are then repeated with the each shortened polypeptide. The PTH derivative formed in each cycle is identified by high pressure liquid chromatography. [6] Average of 20-30 aminoacid from the N-terminal has to be identified for oligonucleotide synthesis. | Figure 2: Protein sequencing using high-performance liquid chromatography
Click here to view |
| Synthesis of the Oligonucleotide Probes | | |
Genomic probe and cDNA probe are derived from cellular material, a third form of probe containing 20 base pairs (bp) synthesized in the laboratory is called oligonucleotide probe. Synthetic oligonucleotide probe can be designed to distinguish normal from abnormal genomic sequences referred to as allele specific oligonucleotide probes. [9],[10]
An oligonucleotide probe containing 20 nucleotides is sufficient to screen a genomic library [Figure 3]. To prepare a specific probe by this method the protein of interest, is usually purified by sequential column chromatography and SDS-PAGE. The purified protein is digested with one or more proteases (e.g. trypsin) into specific peptides. The N-terminal aminoacid sequences of few of these peptides (average of 20 AA) are determined by sequential Edman degradation by using high-performance liquid chromatography. The determined sequences then are analyzed to identify the 6 or 7 aminoacid region that can be encoded by the smallest number of possible DNA sequences. Because of the degeneracy of the genetic codes, the 12 aminoacid sequence shown in [Figure 3] (yellow box) theoretically could be encoded by any of the DNA triplet (red box) below it. The region with the least degeneracy for a sequence of 20 bases (black bracket) is considered. Since the actual sequence of the gene is unknown a degenerate 20 mer probe consisting of a mixture of all the possible 20 base oligonucleotide is included. Based on the genetic code, oligonucleotide probes encoding the determined peptide sequences can be synthesized and radiolabeled. Synthesized oligonucleotide probe will hybridize to the recombinant clone that is perfectly complementary to the actual gene sequence. In the most common approach, a radiolabeled degenerate probe is used to screen a λ cDNA (phage cDNA) library, using the membrane hybridization technique. [10]
| Membrane Hybridization Technique | | |
Membrane hybridization technique is the procedure for screening a λ library. The recombinant λ virions present in plaques on a lawn of Escherichia More Details coli are transferred to a nylon membrane by placing the membrane on the petridish [Figure 4]. Many of the viral particles in each plaque absorb to the surface of the membrane, but many virions remain in the plaques on the surface of nutrient agar in the Petri dish More Details. The original petridish is refrigerated to store the collection of λ clones. The membrane is then incubated in an alkaline solution, which disrupts the virions, releasing and denaturing the encapsulated DNA. Next, the membrane is incubated with a radiolabeled probe under hybridization conditions. Unhybridized probe is washed away, and the filter is subjected to autoradiography. The appearance of a spot on the autoradiogram indicates the presence of a recombinant λ clone containing DNA complementary to the probe.[6]
| Sequence The DNA | | |
Isolated clone can be sequenced using automatic DNA sequencers which is based on the improvement of Sanger's method. [11] Alternative to cloning Polymerase Chain Reaction also can be used to study the DNA sequence. The principle behind this procedure is termination of chain elongation. Where ever there is random incorporation of dideoxynucleotide triphosphates which lacks 3'OH and no phosphodiester bond will be formed, which produce DNA fragment of different length. Each ddNTP is linked with the fluorescent dye that imparts different colors to all the fragments terminating in that nucleotide. DNA fragments of different colors are separated by their respective size in a single electrophoretic gel. The DNA fragments are illuminated with a laser beam. The fluorescent dyes are excited and emit light of specific wavelengths which is recorded by a special recorder. This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by computer. Computer converts the data of emitted light into nucleotide sequence.
| Conclusion | | |
The random primer labeling and Sanger sequencing has shown the way of identifying the specified gene from an unknown protein that is taken out from a cell/tissue or an organ. The extension of the same principle has evolved another powerful technology in the molecular biology namely PCR. If we are able to capitalize this and understand this technique we may be in a position to formulate the gene therapy to tackle the disease processes in oral mucosal lesions.
| References | | |
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| 10. | Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, et al. Complementary DNA sequencing: Expressed sequence tags and human genome project. Science 1991;252:1651-6.
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| 11. | Sanger F. Sequences, sequences, and sequences. Annu Rev Biochem 1988;57:1-28.11.
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Correspondence Address:
B Premalatha
Department of Oral Pathology and Microbiology, Mahatma Gandhi Postgraduate Institute of Dental Sciences, Indira Nagar, Pondicherry
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0970-9290.131145
[Figure 1], [Figure 2], [Figure 3], [Figure 4] |
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