Insight into contemporary molecular diagnostic techniques revolutionizing the specialty of oral maxillofacial pathology and microbiology

Sudip Indu; Vineet Singh Cheema

doi:10.4103/jodd.jodd_13_22

REVIEW ARTICLE

Year : 2022 | Volume : 16 | Issue : 2 | Page : 151-155

Insight into contemporary molecular diagnostic techniques revolutionizing the specialty of oral maxillofacial pathology and microbiology

Sudip Indu¹, Vineet Singh Cheema²
¹ Division of Oral Pathology, Air Force Dental Centre, Kanpur, Uttar Pradesh, India
² 11 Corps Dental Unit, Nalwa Road, Jalandhar Cant, Punjab, India

Date of Submission	17-Jul-2022
Date of Acceptance	01-Sep-2022
Date of Web Publication	21-Dec-2022

Correspondence Address:
Sudip Indu
Division of Oral Pathology, 8 Air Force Dental Centre, Kanpur - 208 004, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jodd.jodd_13_22

Abstract

The specialty of oral maxillofacial pathology and microbiology is currently undergoing a paradigm shift as the present diagnostic protocols continue to upscale itself from the basics of H and E staining procedures, and immunohistochemistry to newer molecular diagnostic techniques such as polymerase chain reaction, comparative genomic hybridization, fluorescence in situ hybridization, and tissue microarray to name a few. In this era of growing numbers of pandemics like COVID-19 affecting the world population, it is imperative for all oral pathologists and microbiologists to keep abreast with these newer diagnostic facilities such that they can be valuable team members along with medical health care providers in the hour of need in encountering severe disease processes such as severe acute respiratory syndrome coronavirus 2, swine flu, and MERS.

Keywords: DNA, DNA sequencing, polymerase chain reaction, RNA, tissue microarray

How to cite this article:
Indu S, Cheema VS. Insight into contemporary molecular diagnostic techniques revolutionizing the specialty of oral maxillofacial pathology and microbiology. J Dent Def Sect. 2022;16:151-5

How to cite this URL:
Indu S, Cheema VS. Insight into contemporary molecular diagnostic techniques revolutionizing the specialty of oral maxillofacial pathology and microbiology. J Dent Def Sect. [serial online] 2022 [cited 2023 Mar 27];16:151-5. Available from: http://www.journaldds.org/text.asp?2022/16/2/151/364513

Introduction

The molecular age histopathologist of today is practicing pathology in a totally different scenario than the preceding generations did. Histopathologists stand, as of now, on the crossroads of a traditional “visible” morphological science and an “invisible” molecular science.

With the rapidly advancing field of biotechnology and molecular biology, a modern histopathologist is expected to be well versed not only in the traditional histopathological techniques but also to keep pace with the ever-expanding frontiers of science and technology.

The analysis of DNA, RNA, and proteins, obtained from diagnostic specimens, has been the area of interest currently revolutionizing the practice of surgical pathology and heralds a new era of diagnostic and prognostic tests that will greatly influence our day today's clinical decision-making.^[1] The diagnosis of cancer and many other diseases is fundamentally based on the microscopic study of cells and tissues. Advances in the analysis of DNA, RNA, and proteins have permitted improved insights into a range of disorders and have led to a better understanding of many diseases.

The extent to which immunopathology and molecular pathologic techniques are used today varies greatly, but it is conceivable that in the near future, many of today's most technically advanced methods of molecular analysis will become a part of standard practice.

All these newer molecular techniques, basically involve the extraction, study, and analysis of the DNA, RNA, and proteins obtained from tissue samples.

Extraction of DNA

High-quality genomic DNA can be successfully prepared or extracted from most cytological and histological samples. DNA can be isolated from formalin-fixed tissue with good results. Neutral-buffered formalin and ethanol are the most commonly used fixatives for molecular techniques.^[2]

The basic steps of DNA extraction from formalin-fixed paraffin-embedded tissue sections tissues include

Cutting thick paraffin sections (50–100 μm)
Deparaffinizing tissue sections
Lysing of the cell using proteinases in the presence of sodium dodecyl sulfate
A high quantity of EDTA is added to inhibit DNase activity
The nucleic acids are recovered by precipitation with alcohol and salt
Only DNA which can be spooled with a glass rod from the final ethanol precipitate is used for subsequent blotting experiments.

Extraction of RNA

The isolation and handling of RNA are more difficult from that of DNA because of the presence of RNase in human and animal tissue. It is advisable to set a special area of the laboratory aside, for RNA work. The two most important denaturing agents used in RNA extraction are guanidine isothiocyanate and guanidine hydrochloride methods.

Laser capture microdissection

Laser capture micro (LCM) is a rapid and reliable technique of procuring only a pure population of cells from tissue sections for microscopic examination.

A histological section is placed on the stage of an inverted microscope and the pathologist selects the area of interest. The basic principle involved in LCM is the preferential adherence of the identified cells to a plastic membrane which is activated by a low-energy infrared laser pulse.^[2] A transparent ethylene-vinyl acetate membrane is positioned above the section. As the operator activates a laser beam, the film above the targeted area melts and the long chain polymers of the film surround and tightly hold the cells. The final evaluation and molecular techniques are then carried out on these selected cells.

There is only minimal sample loss and the selected cells can be made to undergo various staining procedures and molecular techniques.

Tissue Microarray

Tissue microarray (TMA) is a method of relocating tissue sections from conventional histologic paraffin blocks such that sections from multiple patients or blocks can be seen on the same slide. In simple terms, TMAs are multiple tissues in a single paraffin block.^[3] The TMA consists of cylindrical paraffin-embedded tissue cores, which are acquired from primary tissue blocks. The donor block is a standard tissue block. A morphological representative area of interest is identified within the histological slide and then identified subsequently on the donor block. The representative tissue cores are removed from the donor block and transferred to the recipient paraffin block, using custom-made Beecher instruments.

Using a precise spacing pattern, tissues are inserted at high density, with up to 1000 tissue cores in a single paraffin block. Sections from this block are now cut with a microtome and are placed onto standard glass slides that can then be used for further analysis.

Depending on the overall depth of tissue remaining in the donor blocks, tissue arrays can generate between 100 and 500 sections. Once constructed, TMAs can be used with a wide range of techniques including histochemical staining, immunohistochemical/immunofluorescent staining, or in situ hybridization (ISH) for either DNA or mRNA.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) provides a convenient and efficient means of making millions of copies of a short DNA sequence. Heating–cooling cycles are used to denature DNA and then build new copies of a specific, primer-bound sequence.

Polymerase chain reaction process

Genomic DNA is first heated and denatured at a temperature of 95°C to form single strands, in the denaturation phase. Next, in the annealing phase, the DNA is cooled to 55°C, allowed hybridization with primer sequences that flanks the region of interest. Then the reaction is heated to an intermediate primer extension temperature (72°C), in which DNA polymerase adds free bases in the 3'direction along each single strand. Now, blunt-ended DNA fragments are formed, which in turn form the template for the next heating and cooling cycle. Repeated cycling produces a large number of DNA fragments bounded on each end by the primer sequence.^[4]

Components of typical polymerase chain reaction

Template (Target sequence)
Primers
dNTP's (free nucleotides)
Taq DNA polymerase
Buffer, containing magnesium
Gelatin.

Applications of polymerase chain reaction

Because of its speed and easy usage, it is widely used in the following conditions-

Diagnosis of infectious diseases and malignancies associated with these microorganisms. For example., Epstein–Barr virus, human papillomaviruses (HPV), and human herpes virus-8 infections
To assess genetic variation and diagnosis of chromosomal disorders, e.g. sickle cell anemia and hemophilia
Forensic pathologies such as sex determination and paternal testing
Study of oncogenes in tumor biology.

Modifications of polymerase chain reaction

Polymerase chain reaction advantages

Extremely small quantity of DNA can be utilized for further amplification
Fast, automated, and multiple copies can be made
Safe, nonradioactive probes can be used, e.g. biotin.

Polymerase chain reaction Disadvantages

Very technique sensitive
Chances of contamination are high
Exhaustion of primers can be a major hindrance
Difficult to amplify larger base pairs (>300 base pairs) of normal sequences
Specificity is limited to many interrelated factors such as NTP, primer size, buffer salt, and annealing temperature
No proofreading properties are available as nucleotide addition errors can take place.

A few of the most commonly used modifications of the PCR are here as under.

Reverse Transcription-Polymerase Chain Reaction

It is also known as reverse transcriptase PCR, it is used for the identification of the presence of RNA virus in general and for molecular cytogenetic analysis of chromosomal translocations. A complementary DNA sequence is made from the extracted RNA which is further amplified and analyzed. One major difference between the common PCR techniques is the use of Thermus thermophillus enzyme, rather than the DNA Taq Polymerase enzyme. This enzyme has an inert reverse transcriptase enzyme, which helps in direct conversion into cDNA sequences.^[5]

Nested polymerase chain reaction

It refers to the use of two sequential PCR reactions with the primers used for the second PCR reaction, internal to those used for the initial PCR reaction. It increases the sensitivity and specificity of the reaction.

In situ polymerase chain reaction

PCR is carried out within intact cells, tissue sections, and cytological specimens. The amplified product is seen in a microscope rather than gel/membrane by the use of labeled nucleotides during the actual PCR. It is helpful in the detection of viral infections such as HIV and HPV.

Competitive polymerase chain reaction

It is done to semi-quantify the number of abnormal genes present and is applied to the detection of minimal disease in neoplasia. Two sets of primers are used one is patient-derived DNA and the other is artificially made DNA in the same reaction.

Hybridization methods

Hybridization refers to the pairing of complementary RNA or DNA strands to produce a double-stranded nucleic acid. The nucleotide base pair relationship is so specific that strands cannot anneal unless the respective nucleotide strand sequences are complementary. All hybridization methods use a radiolabeled or fluorescence-labeled DNA or RNA probe that binds to the target DNA or RNA of interest, permitting visualization. The target nucleic acids can either be immobilized in a membrane (“blotting”) or examined in tissue sections (in situ).

Southern blotting

A widely used method for analyzing the structure of DNA was described by Southern.^[6] This involves the transfer or blotting of DNA fragments onto a membrane. The specific DNA fragments which have been separated by gel electrophoresis, and transferred to a nylon or nitrocellulose membrane can be identified by hybridizing the membrane with labeled complementary DNA or RNA probes, followed by detection of the label on an X-ray film by autoradiography.

Northern blotting, Western blotting, and Eastern blotting

The separation and identification of RNA in the above fashion is referred to as northern blotting, western blotting for analysis of proteins, and the recently added terminology of eastern blotting for the study of posttranslational modification proteins.

In Situ Hybridization

ISH are techniques used to examine DNA and RNA in thin normal sections in their normal topographic surroundings. The technique allows localization of nucleic acid sequence, viral RNA, DNA or cellular RNA, or chromosomal DNA. It involves the specific hybridization of a labeled nucleic acid probe to complementary target sequences in tissue, followed by visualization of the location of the probe.

Applications of In situ hybridization

Can be used in many areas of biology, medicine, embryology, and microbiology
Valuable in genetic studies, allowing gene expression studies
To identify viral sequences in viral infected tissue
Provide valuable insights into pathogenic mechanisms of viral infections.^[7],[8]

ISH and immunohistochemistry (IHC) have provided new methods for the identification and study of many viruses. Both have good sensitivity in identifying viral DNA (ISH) and viral proteins (IHC). However, ISH scores over IHC in cases where no antigens are present in the tissue section or when commercially prepared antisera are unavailable.

DNA Microarrays

A DNA microarray, also commonly known as a DNA chip, is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface such as glass, plastic, or silicon chip by covalent attachment to a chemical matrix. Each DNA spot contains a specific DNA sequence, known as probes.

Hybridization of a probe to multiple defined genomic DNA's/cDNA's which are robotically printed onto specific locations of the chip.^[9] Probes and its hybridization with cDNA from reverse transcription of tumor mRNA, labeled with a fluorescent marker, produce a hybridization signal in the form of red, green, and yellow fluorescent emissions which are scanned by a reader consisting of argon lasers and confocal microscopy. The expression intensity of 1000's of different genes can be compared to expression in normal tissues at a single time.

The cDNA microarray can be helpful in studying the mRNA or gene expression profiling in tumors and normal tissues. It can be utilized in comparative genomic hybridization (CGH) in assessing genome content in different cells or closely related organisms.

Comparative genomic hybridization

CGH measures DNA copy number differences between a reference genome and a test genome. Early CGH experiments and the DNA targets were hybridized to metaphase chromosome spreads in fluorescence ISH assays. In simple terms, CGH permits the development of a detailed map of chromosomal differences between normal and tumor cells by detecting increases (amplifications) or decreases (deletions) of segments of DNA.

DNA sequencing

The primary goal of any genetic study is to determine the actual array of DNA base pairs that composes a gene or part of a gene. The DNA sequence can indicate a great deal of information about the nature of a specific mutation, which forms the base of many tumor studies.

The dideoxy method of DNA sequencing, invented by Sanger, makes use of chair-terminating dideoxynucleotides. Chemically, they are very similar to the deoxynucleotides, but with a missing hydroxyl group.^[4] The absence of the hydroxyl group prevents the formation of new phosphodiester bonds with the free DNA bases. Hence, once these dideoxynucleotides are added to a growing DNA helix, no additional nucleotides can be incorporated. Four different dideoxy nucleotides (A, G, C, and T) are used.^[10]

The basic ingredients of the mixture are

Single-stranded DNA whose sequence is needed to be determined mixed with radioactive-labeled primers
DNA polymerase
Deoxynucleotide triphosphates
One type of dideoxynucleotides.

The cycle proceeds as in a normal PCR with complementary base pairing, the addition of free bases, and finally, the addition PCR of the dideoxynucleotide. This addition of either the nucleotides/dideoxynucleotides is a random process. Hence, DNA fragments of varying fragments, each ending with the different dideoxynucleotides are separated by gel electrophoresis. Four different sequencing reactions are run, each with a different base and finally, on the gel electrophoresis, each unique DNA sequence can be analyzed by observing the bands. More recently, to compensate for the slow, laborious, error-prone process of DNA sequencing, automated DNA sequencing using fluorescent labels, and a laser detection system has greatly increased the speed and efficiency of the sequencing process.

Detection of variation at DNA level

It is estimated that humans vary at approximately 1/300 to 1/500 base pairs. It is possible that 10 million polymorphisms may exist among 3 million base pairs, composing the human genome.

Restriction Fragment Length Polymorphism

A bacterial enzyme called restriction endonucleases prevents the entry of foreign DNA into bacterium by cleaving the DNA at specific recognized sites called restriction sites, which produces DNA restriction fragments. For example, Escherichia coli produces a restriction enzyme Eco RI, which recognizes the DNA sequence GAATTC only and cleaves the sequence between G and A.

The gel electrophoresis separates these DNA fragments according to their size of the DNA fragments. Further, the DNA is denatured and transferred to a solid membrane, hybridized with a radioactive probe, and exposed to X-ray film by autoradiography, revealing specific bands of DNA fragments. These are the polymorphisms revealed by variations in the length of the restriction fragments.

Variable Number of Tandem Repeats

This particular technique utilizes the minisatellites (same DNA sequence repeated over and over again) that exist throughout the genome. The genetic variation measured here is the number of repeats in a given region, which varies from individual to individual.

The only difference in the technical aspects in comparison to the restriction fragment length polymorphism (RFLP) is the special probes which hybridize only to minisatellite regions. Thus, a variable number of tandem repeats (VNTRs) are a form of RFLP which arise from variation in the number of tandem repeats in a DNA region.^[4]

Microsatellite repeat polymorphisms

A microsatellite repeat is a substantially smaller nucleotide (2/3/4 base pair in length) than a minisatellite. They are more abundant than VNTRs, more evenly distributed in the genome, and easier to assay in the laboratory. Thus, they are the polymorphisms of choice in most gene mapping studies and forensic applications such as paternity testing and identification of criminal suspects.^[4]

Conclusion

The various molecular techniques mentioned above have permitted improved insights into a range of disorders and have led to improvements in the understanding and classification of many diseases. This article has described only the most important molecular, tools that are currently being used or will be used in diagnostic pathology, as well as their applications. In the future, the pathologist will continue to play a central role in diagnosis and it is conceivable that the armamentarium of diagnostic tests will include many of these advances. Thus, these changes likely will improve the understanding of diseases that affect the head and neck and the ability of the pathologist to render a diagnosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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