INTRODUCTION:

The extensive progress in nucleic acid technology has been created a profound knowledge. The analytical techniques such as DNA sequencing, Polymerase chain reaction, Gel electrophoresis, Blotting techniques, DNA fingerprinting and Heteroduplex analysis have created a vast amount of genetic information. The techniques also provided prediction stages for aligning functions which may correlated with the genetic disorder or diseases¹. The fame of nucleic acid in entire areas of Biosciences calls for the prompt development of successful methods for their extraction, isolation, quantification and structural analysis².

The analysis of nucleic acid is critical part in biological sciences. Due to structural complexity, biological scientists need to focus on analytical methods and criteria use during analysis. Some of the external environmental factors may impose a defect or even sometimes internal factors may deteriorate the findings. The high-resolution separation techniques transformed the way of nucleic acid analysis³. In the modern biological experimentation, analysis the variation in the sequence and abundance of nucleic acid support⁴.

Every biological species in nature consist of nucleic acid structures. Upon breaking the nucleic acid by using chemical or enzymatic approach produces phosphoric acid, sugars and a mixture of organic bases (purine and pyrimidine)⁵. Nucleic acid consisted of abundant genetic information and transported across cells by governing the process of protein synthesis⁶. The two nucleic acid types are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleic acid compositions decide the inherited traits for every living creature^7,8. DNA is the principle pattern for life and comprised of genetic material in every free-animate organism. DNA is also found in most of the viruses by molecule contributing its genetic behavior. The genetic material of the virus look likes particles to help in designing outer protein shield⁹.

The DNA represents a subset of Genome inside the eukaryotes. In human, DNA located in nucleus and consists of 23 pairs of chromosomes. An additional mitochondrial DNA is also represented maternal family ties and converts energy required for cell functioning. Mitochondrial DNA is circular differentiating from the nuclear DNA with linear nature. In human, genomes are diploid and inherited from each parent. A complete and performing diploid genome is essential for normal development and to sustain life¹⁰. The researchers are working to develop robust methods for nucleic acid identification. In recent times, nanobased techniques are improvising the results with reducing the time of analysis. The scope of research is expanded towards, disease identification, staging, identification of direct effect of medicines on genes, forensic utility and few of the environmental applications. Many analytical techniques developed till dates are categorized as essential secondary detection techniques¹¹.

This review paper intended to provide principles, methodology and techniques of nucleic acid analysis. The chapter gives in depth knowledge of identification and interaction with the substrate for the analysis. The interaction of nucleic acid and analyte is most important during sequencing steps. The interaction predicts exact differentiation among the amino acid’s chains in the nucleic acid.

Nucleic acid:

The nucleic acid is the polymers of nucleotides¹². The nucleic acid assembly consists of monomeric units and the structure will forms through the bridge in between individual units¹³. Nucleic acid is large biopolymer consist of longer linear chains of amino acids. The purines and pyrimidine units are attached and forms helical structure. It carry huge amount of genetic information and consist of all forms of life. Single form of biomolecule abbreviates to represent the morphology, structure, shape, size, etc, of all living things¹⁴.

The nucleic acids are hydrolyzed in presence of enzymes like ribonucleases and deoxyribonucleases¹⁵.

Compositions of Nucleic acid:

The nucleic acid is chemically composed of carbon, oxygen, hydrogen, phosphorus, and nitrogen.¹⁹. Nucleic acid contains nitrogen about 15-16% and phosphorus about 9-10%. On hydrolysis of nucleic acid in the presence of an enzyme or heating with dilute acids or alkali yields Nucleotide¹³.

The composition of nucleic acid discussed in following section⁹.

1. Nucleotide and Nucleoside:

a) Sugar [Ribose, Deoxyribose]

b) Purine base [Guanine, Adenine]

c) Pyrimidine base [Cytosine, Thymine, and Uracil]

2. Phosphoric acid or Phosphorus group

Nucleotide:

A nucleotide is consisting of the nitrogenous base, pentose sugar, and Phosphate group. Nucleotides have complete functions inside the living cells, apart from existing in building blocks or monomeric units in the nucleic acids (DNA and RNA)⁶. These also provide part of as a structural component of some coenzymes of Beta complex vitamins (e.g. FAD, NAD+), is involved in the energy reaction of cells as well as regulate metabolic process⁷.

Structure of Nucleotides:

Nucleotides are contained the following three portion -

1) Phosphoric acid or phosphate

2) Sugar

3) Heterocyclic Bases or Nitrogenous bases. (Purine and Pyrimidines)

Phosphate is the link in the 5^th position at the carbon of the pentose sugar structure of nucleotides.⁸

1) Phosphoric acid or Phosphate:

The total phosphate present in Nucleotide may be one or two or three and the Phosphate remnant is attached in the 5^thcarbon of the pentose sugar structure of nucleotides.¹⁶

2) Sugars:

A nucleotide contains five carbon monosaccharides (Pentoses). The RNA contains sugars as Ribose and DNA contain sugar as Deoxyribose. They differentiated from the structure in at carbon no. 2 Deoxyribose has one oxygen less than the as to Carbon no. 2. The sugars consist of the furan as heterocyclic rings.^{9, 20}

3) Nitrogenous bases:

Nitrogenous bases are present in nucleotides an aromatic heterocyclic compounds.

The two kinds of nitrogenous bases are present in Nucleotides.

A) purines B) Pyrimidine

The general structure consists of purines and pyrimidine’s subunits. According to well accepted nomenclature system the purine subunit numbered in opposite direction while pyrimidine subunits numbered in clockwise direction. The consecutive number of each subunit represents base structure⁷. DNA consist of the purine bases are namely Adenine (A) and Guanine (G) and Pyrimidine bases are namely as Cytosine (C), Thymine (T). RNA consist of the purines contains Adenine (A), Guanine (G) and Pyrimidine bases are namely as Cytosine (C), Uracil (U).¹⁵

Nucleoside:

It consists of purine or Pyrimidines bases and pentose sugar (Ribose / Deoxyribose)⁹

The simple Nucleoside is defined as- Nucleoside= Ribose / Deoxyribose + Bases. The synthetic compounds of nucleoside are involving in inhibiting the growth of cancer cells or certain viruses.¹⁹

Types of Nucleic acid²¹

These are the two main class of nucleic acid.

1) DNA (deoxyribonucleic acid)

2) RNA (ribonucleic acid)

DNA:

DNA is a polymer of deoxyribonucleotides. It is found in Chromosomes, mitochondria, and chloroplasts. It carries the genetic information from one organism to another¹⁷. The information in DNA is stored as code (made up of A, G, C, and T).²⁶ The structure of DNA shows a long-chain polymer of nucleotides comprise -Deoxyribose, phosphate group, and organic bases such as Adenine, Guanine (Purines), and Cytosine, Thymine (Pyrimidines).¹⁶

DNA is a twofold helix structure with two strands which gives stepping stool like shape with base pairs²¹. In 1953 Watson and crick postulate a 3-D model of a DNA helix structure. Presently, the other two fold helical structures also postulated based on modern biological methods. The modern biological methods attempt to provide newer achievements in DNA structure. DNA additionally exists in a certain strange structure like Bent DNA, Triple stranded DNA, and four-stranded DNA.²⁵.

RNA:

RNA is the polymer of ribonucleotides joins by 3'-5' phosphodiester bridges.¹⁴while RNA has undisputed looks similar to DNA structure. While many differentiations or varieties available with RNA structure.

1) Pentose sugar-Ribose

2) Pyrimidines base-uracil instead of thymine.

3) Single strand.

4) Prone to alkali hydrolysis.

5) Orcinol color reaction -RNA are histological point out by utilizing of orcinol.^17,22

The three types of RNAs are Messenger RNA(mRNA), Ribosomal RNA(rRNA), Transfer RNA(tRNA).²³ Along with these other 3 more RNA types are available in the cells such as Heterogeneous nuclear RNA, small nucleolar, small nuclear RNA, and small cytoplasmic RNA.²⁴ The RNA is preliminary synthesized from DNA and is principally involve in protein synthesis.⁷. Messenger RNA is synthesized in the nucleus & mRNA which enters the cytoplasm to initiate protein synthesis.²⁵. The ribosome is an initiator for protein synthesis. The eukaryotic ribosomes are made out of two significant nucleoprotein complexes -60s subunit and 40s subunits. The rRNA is found in the ribosomes and a total of 80% RNA found in the cell.⁹. Transfer RNA is the smallest of all 3 types of RNA. The tRNA possess about 75-95 nucleotide subunits. The tRNA is an basic part in the translation process.¹⁷

Analytical Techniques use of Nucleic acid analysis

1. Gel electrophoresis:

Gel electrophoresis is a technique applied for the isolation & analysis of DNA, RNA, or their fragments on the basis of their size and charge.²⁶.

In this process, the gel used an anti-convective media / sieving media throughout electrophoresis. Gel suppresses the thermal convection done by use of the electric field. gels are also serving to maintain the finished separation so that after electrophoresis stain can be applied. The gel is a colloid in solid form nature. The gel is used to support media is electrically neutral²⁷.

The Buffering system is essential when charged molecules are electrophoresis through a separation medium. The most frequently used buffer Tris-acetate EDTA and Tris-Borate EDTA, Tris-citrate EDTA buffer, Tris-Glycine Buffer, and phosphate buffer.²⁸ After the electrophoresis is done, the molecules present in the gel can be stained to make them visible. Nucleic acid can be stained after electrophoresis separation. soaking the gel in a solution of ethidium bromide. Ethidium bromide/silver or coomassic blue used the next process. The visualization of stained DNA in gels using the ultraviolet lightbox.²⁹

Principle:

The add small sample to be isolated in wells of the agarose/ polyacrylamide gel & by using an electric current, permits the molecules to pass through the matrix at various rates towards the anode, if negatively charged or toward the cathode, if positively charged they move through the gel, the bigger atom will be held up as they attempt to go through pores of the gel, while the littler particles will be blocked less and move quicker. These outcomes in a detachment by size, with the bigger atom closer the well & the littler particle further away.²⁸

Application:

Isolation and analysis of DNA, RNA, Protein molecules, an amino acid.³⁰

2. Blotting techniques-

Blotting techniques are generally utilized analytical tools for the particular identification of desired DNA or RNA fragments from hundreds of molecules. Blotting refers to the methods of immobilization of the test nucleic acids on a solid support (nitrocellulose or nylon membrane). The blotted nucleic acid is then utilized as targets in the hybridization tests for their particular recognition.⁵ The most frequently use blotting techniques are Southern and Northern blotting.

The southern blotting is named after the scientist Ed Southern in 1975. He created it. It is a technique routinely utilized for the detection of a particular DNA sequence in DNA substance.³¹

Northern blotting is a method for the particular identification of RNA molecules. James Alwine and George stark develop northern blotting method at Standford University.³²

Principle:

The method includes the exchange of electrophoresis separated DNA fragments to a transfer layer which is usually nitrocellulose and subsequent detection of the target DNA fragments by probe hybridization. Hybridization is the way of formation of a double strand DNA into a single-stranded DNA probe and a single-stranded DNA since the probe and target DNA are complementary to one another, the reaction is explicit which helps in the detection of the specific DNA fragments.³³.

Figure 2. Schematic representations of Blotting techniques.

Table 1 Application of Blotting techniques

Sr. No.

Blotting Technique

Application

Ref.

Southern Blotting

1. Identify specific DNA.

2. Mutations detection and Gene arrangements.

3. Prognosis of Cancer.

Northern Blotting

1. Identification of RNA.

2. Detection of mRNA transcript size.

3. Study degradation& half life of RNA.

3. Denaturing gradient gel electrophoresis (DGGE):

Denaturing gradient Gel electrophoresis is a technique utilizing for the isolation of DNA fragments as need be to their mobilities under progressively denaturing conditions usually increasing (formamide/urea Concentration).³⁵

Principle:

Little substance of DNA or RNA is added to an electrophoresis gel that contains a denaturing agent.³⁶ The denaturing gel induces melting of the DNA at different stages and DNA spreads through the gel and makes single-stranded branches. This branching design is made conceivably by the ‘GC clamp’, a located of high GC contact inside the PCR primers. This change in relocation rate makes a banding pattern like that seen for fluctuating size fragments in gel electrophoresis. To Contrast, these bands with known sequences would then be able utilized to recognize the species present in the substance.³⁷

Application:

· Entirely appropriate methods for the identification of Novel or obscure organisms.³⁷

· To Analyze the DNA from microbial communities without cultivation.

· Analysis of RNA.[38]

4. Temperature gradient Gel electrophoresis (TGGE):

TGGE is an amazing method for the isolation of Nucleic acids.³⁹ Temperature gradient Gel electrophoresis was first developed by Lerman and Andersen of Georgia Utilizing a Beryllium oxide plate as a thermal diffuser.⁴⁰

Principle:

A little sample of DNA / RNA is added to an electrophoresis gel in which temperature gradient is utilized to denature molecules as they travel through either acrylamide or agarose gel. The DNA or RNA changes the structure with increasing temperature.³⁶ The two strands will isolate double-stranded molecules into a single-stranded molecule. Mobility of DNA molecule through the PCR amplification and detected.⁴¹

Application⁴²:

· To analysis the Nucleic acid.

· To analyze the Protein-DNA complex.

5. Polymerase Chain Reaction (PCR):

The PCR is a typical technique practiced in hereditary research centers since it is a fundamental necessity for a hereditary or molecular lab.

In 1983, Kary Mulis reported the method of in vitro gene amplification & further called it a Polymerase chain reaction. the PCR is a temperature- dependant cycle of DNA amplification. The machine utilizes in the PCR techniques is called a thermocycler.⁴³ Denaturation, annealing, and extension of DNA happen at various temperatures. The most generally utilized target Nucleic acid amplification technique is the PCR.

Template DNA (Plasmid DNA, bacterial DNA, and cDNA), PCR primers, dNTPs (Deoxyncleotide triphosphates), PCR Buffer, &Taq DNA polymerase are the main components of PCR reaction^.44

Principle:

The principle of Polymerase chain reaction based on the thermocycling reaction categorized into three parts-

· Denaturation- The dsDNA is denature at high temperature and it is converted into single-stranded DNA. In this step Hydrogen bonds present between two DNA strands is break during denaturation process.

· Annealing- In this step primer binds or anneals to its exact complementary sequence on DNA.During annealing step the primer provides a site for the initiation of synthesis.

· Extension- 3’ ends of the primer uses the Taq DNA polymerase & start synthesis of DNA by adding nucleotides to the growing of DNA strands.⁴⁵

Figure 5. Schematic representation of PCR.

Application⁴⁵:

· PCR can amplify a single DNA & RNA molecule from a complex mixture.

· Investigation of Gene expression.

· In screening for human genetic disease.

· PCR is sensitive and can amplify small amounts of DNA of specified bacteria and viruses.

· PCR uses in Forensic medicine.

6. Real-time Polymerase Chain Reaction:

It is a method utilize to screen the advancement of a Polymerase chain reaction in real-time at the subsequently, a moderately little quantity of PCR product macromolecules can be measured. It depends on the fluorescence create by a reporting molecule and increases as the reaction continues.⁴⁶

Real-time PCR also known as a Quantitative polymerase chain reaction, which is laboratory techniques of molecules or biology depends on a polymerase chain reaction.⁴⁷

Principle:

This is the same principle of amplification of PCR is used in real-time PCR. But rather than of visualization the band on a gel towards the finish of the reaction, the processed observed in 'real-time.’⁴⁷This reaction is put into a real-time Polymerase chain reactio machine that observe the reaction happen with a camera or detection of the fluorescence produced by the excited fluorophore.⁴⁸

Figure 6. Schematic representation of Real -time PCR.

Application⁴⁸:

· Disease diagnosis and management- viral quantification.

· Gene expression analysis- Cancer research and Drug Research.

· Animal and plant breeding-Gene copy number.

7. DNA fingerprinting:

DNA fingerprinting or DNA profiling is a method used to decide the Nucleotide sequence at the aspecific aspect of the DNA that is universal in all human beings.⁴⁹

The method of DNA fingerprinting was discovered by Sir Alec Jeffery at the University of Leicester in 1985.⁵⁰

Principle:

The DNA molecule is first broken with the help of enzyme restriction endonucleases that further formation in DNA fragments are isolated as per the size by gel electrophoresis.⁴⁹ They are treated with alkaline chemicals to breaks into single-stranded DNA and move onto a nylon membrane. Single strands bind with radioactive probes. the X-ray film shows bands.⁵¹

Application^50,52:

· Forensic science-Biological material used for DNA profiling. It is useful in solving like murder and rape.

· Diagnosis of inherited Disorders.

· Detection of somatic mutations or cancer.

· Sex determination.

· Help Diagnose disorders in both prenatal and newborn babies.

· Personal Identification.

8. Molecular markers or DNA markers:

A molecular marker is a DNA sequence that is promptly detected, and whose inheritance can easily be observed. Explicit fragments of DNA that can be recognized inside the entire genome.⁵³

A molecular marker is an overall test that permits the detection of the sequence contrast between two or more individuals.⁵⁴

Principle:

DNA molecule with specific restriction enzyme break DNA into DNA fragments. They is isolated in gel electrophoresis. Capillary action gets on alkaline chemical upward through the gel and a sheet of nitrocellulose paper is laid on the head of it.⁵³ Putting the DNA to the paper and denaturing it simultaneously. The single strands DNA adhere to the paper, situated in bands precisely as on the gel. The paper blot is exposed to a chemical containing radioactively labeled the probe and afterward visualize bands in autoradiography.⁵⁵

Application⁵⁴:

· The measure of Genetic Diversity.

· Genotype Pyramiding and introgression.

· Fingerprinting.

· Genotype solution.

· Identification of Genotype.

9. Heteroduplex analysis:

Heteroduplex analysis is a technique in biochemistry used to detect point mutations in DNA since 1992.⁵⁶ This technique of investigation relies on the way that Heteroduplexes shows diminished versatility comparative with the Homoduplex DNA. In a mixture of wild-type and mutant amplified DNA, Heteroduplex is shaped in wild-type alleles.⁵⁷

Principle:

The principle behind this method is that DNA strands can be isolated from one another upon denaturation and reannealing to form Heteroduplexes and Homoduplexes.⁵⁶ Furthermore, the heteroduplexes DNA spreads move slower on a polyacrylamide gel. When comparison to homoduplex DNA. heteroduplexes analysis in PCR amplification.⁵⁷

Figure 9. Schematic representation of Heteroduplex analysis.

Application⁵⁸:

1. Diagnostics tools for detecting sequence variations.

2. Used for mutation detection in genomic DNA.

10. Single-strand conformation polymorphism (SSCP):

Single-stranded conformation polymorphism analysis is a generally utilized screening technique that permits you to recognize diverse genomic variants in an enormous number of samples and a wide range of organisms, from microorganisms to humans.⁵⁹

SSCP utilize to be an approach to find out new DNA polymorphism besides DNA sequencing.⁶⁰

Principle:

This technique is based on the principles of amplified DNA is first denaturated and afterward subject to non-denaturating gel electrophoresis.⁶¹ That utilization double-stranded DNA which is changed over to single Stranded DNA is an undeniably denaturating physical condition during gel electrophoresis and analysis to detect sequence variations through electrophoretic mobility differences. DNA that contains a sequence mutation has a quantifiable. Mobility difference compared to wild type DNA.⁶²

Application⁶¹

· SSCP is a diagnosis appliance in molecular Biology.

· It is used in genotyping to detect homozygous and heterozygous individual different allelic states.

· SSCP is used to detect variation in different strains of a virus.

11. DNA sequencing:

This technique is originally developed by Fredrick sanger in 1975, most DNA sequencing that happens in medical and research labs today is performed using sequence employing in varieties of sanger method.⁶³

Figure 10. Schematic representation of SSCP.

The term DNA sequencing refers to the sequencing technique for deciding the order of the nucleotide bases in the DNA molecule.⁶⁴ DNA sequencing, and in various applied fields such as forensic, Diagnostic, Biotechnology, and Biological systematic.⁶⁵ Specialists still, however, should have the option to decide the arrangement of bases in DNA that makes up the human genome.

DNA sequencing is the two types- sanger sequencing and Maxam-Glibert method.⁶⁶ Fredrick Sanger and associates developed sanger sequencing technique and he got Nobel Prizein 1980.⁶⁷ Maxam-Gilberts sequencing is a technique of DNA sequencing developed by Allan Maxam & Walter Gilbert (1976-1977).⁶³

Application⁶⁶:

· Understanding and function of specific sequence and the sequence responsible for any disease.

· It helps with comparative DNA sequence study we can detect any mutation.

· DNA fingerprinting.

· Knowing the whole genome sequence.

· Forensic science for identifying the particular individual.

· Play a vital role in agriculture fields.

· Medical research in detection of hereditary or acquired disease.

Future Projection:

Nucleic acid analysis is most vital and fundamental technique of analysis. In the recent years of developments many viruses or bacterial infections are emerging. The therapeutic output of such virulent genes is very tedious job for researchers. Certain life threatening events have been faced by many countries. The newly developed viruses are mutating their structure and Modulate accordingly in environment. The remarkable changes noted from SARs and COVID- 19, etc. Lakhs to millions of death recorded and no specific therapy is available for Effective treatment.

Whereas diagnostic system currently utilized is based on analysis of Genomic component. The nucleic acid analysis techniques are taking new shape of Theranostic approach that simultaneously detect and deliver the therapeutic agent at the targeted site. Although, nucleic acid-based analysis is also reshaping the molecular pathways along with specificity towards analyte. The Nucleic acid projections are so much important to trace the modifications occurs during mutations and probable approach utilize for therapy. Computational based therapeutic approaches are also considering the basic data sets of nucleic acids for analyzing specific targets.

Nucleic acid-based analysis approaches systemized the methodologies available for diagnosis and treatment. Whereas nucleic acid analysis techniques are relevant for future developments in sequence-based analysis of Infectious diseases.

CONCLUSION:

In Conclusion, the paper provides a structural knowledge about nucleic acid. From basic structure to interlinking analysis was elaborated. The basic highlights of nucleic acid are important for revisiting the possibilities from biological sciences. The structure-based analysis approach gives more insights for the development of laboratory methods. The methods highlighted within this section are focusing on general laboratory protocols suitable the routine analysis. The approaches covered impediment over the difficult and time-consuming methods. The protocols highlighted were useful for commercial laboratories as well as removing the barriers during analysis. The implied structural methods are covered to get the extent of knowledge during analysis.

REFERENCE:

1. Griffiths AJF, Miller JH, Suzuki DT. An Introduction to genetic analysis. Kolkata: W.H. Treman and Co. ;1996.

2. Walkar JM. Methods in Molecular Biology. Clifton:Human press;1988.

3. Matsuo T., Caprioli RM, Gross ML. Biological mass spectroscopy: Present and future. Chichester: John Willey press; 1994.

4. Keer JT, Birch L. Essential of Nucleic acid analysis. Cambridge: RSC publishing;2008.

5. Dr. Satyanarayana U, Dr. Chakrapani U. Biochemistry. 3rd ed. Kolkata: Book and Allied Publisher; 2010.

6. Chawala Rajinder, Malwalli Tarek H. Text book of Medicinal Biochemistry. 1st ed. New Delhi: United India Periodicals; 2012.

7. Chatterjea MN, Shinde Rana. Text Book Medicinal Biochemistry. 8th ed. New Delhi: Jaypee Brothers Medical Publisher; 2012.

8. Vasudevan DM, S. Shreekumari, Vaidaynathan Kannan. Textbook of Biochemistry for medical students. 6th ed. New Delhi:Jaypee brothers medical Publisher; 2011.

9. Minchin Steve, Lodge Julia. Understanding biochemistry:structure and function of nucleic acid. Essays in Biochem. 2018; 63:433-456.

10. Laroy J Kricka. Nucleic acid detection technologies.clin chem .1999; 45(4): 453-458.

11. Baynes John W, Dominiczak Marck H. Medical Biochemistry. 5th ed. Edinburgh: Elsevier; 2018.

12. Puri Dinesh. Textbook of Medicinal Biochemistry. 6th ed. USA: Springer; 2020.

13. Lodish H. MolecularcellBiology.4thed.NewYork:W.H.Freeman;1999.

14. Gesteland RF. The RNAworld.2nd.NewYork: Coldspring Harbor Laboratory Press;1992.

15. Saenger W. Principles of Nucleic acid structure.1^st ed. New York: Springer-verlag;1992.

16. Chargraff E. Structure and function of nucleic acid as cell constituents.Q.J. Microsc Sci.1951;10: 654-659.

17. Uhlenbeck OC. Pardi A. Feigon J. RNA structure comes of age.Cell.1997;90:833-840.

18. Daisuke M.Yoshi, Simenel Friendrich C, Marky Luis A. Synthesis and application of Nucleic acids.J. Nucleacid.2011; 2011:1-2.

19. Watson JD, CrickFH,. Molecular structure of Nucleic acids, Nature.1953; 171(4356):737-738.

20. Wilkins MH, Stokes AR, Wilson HR. Molecular structure of deoxypentose nucleic acid.Nature.1953; 171:738-740.

21. Arboleda Valerie A, Xian Rena R.An over view of DNA analytical methods. Methin Mol Biol.2019;1897: 385-402.

22. Doherty EA, Doudna JA. Ribozyme structur s and mechanisms. Rev. Biophys. Biomol. Stru. 2001; 30:457-475.

23. Breaker Ronald R. Joyce Gerald F. The expanding view of RNA and DNA function. Chem Biol. 2004; 21(9) :1059-1065.

24. Franklin RE. The structure of DNA and RNA. New York: Science of America; 2002.

25. Alneida Maria J., Reis Rui M, Calin George A. microRNA history: Discovery, recent application and next frontiers. Springer. 2008; 22:1028-1042.

26. Kryndushkin DST, Avanesyan MD. Yeast prion aggregates are formed by smalls up 35polymers fragments by HSP104. J.Bio Chem.2003; 278(49): 636-43.

27. AryalSager. Agarose Gelelectrophoresis [Internet]. 2018. Availableform:https://microbe-notes.Com/Agarose-gel-electrophoresis/.

28. Adamsan A.W. A hand book forgel electrophoresis. Maine: Cambrex; 2004.

29. Dr.Sharma BK.Instrumental methods of chemical analysis.1^st ed.Meerut: Krishna Prakashan;2002.

30. Aajj C. Borst P.The gel electrophoresis of DNA.Biochem Biophys Acta.1972;2:192-200.

31. Blot (Biology) [Internet]. Available form:https://en.m.wikipedia.org/wiki/blot.(Biology).

32. Koscianska Edyta, Roslan Julia, starga Kozyzosiak Wlodzimerz J.Northern blotting analysis of micro RNA pair precursor or and RNA a interference triggers. BMC Mol Bio.2011;14: 12-14.

33. Aryad Sagar. Southernblot-Principle steps and application Internet]. 2018. Available form:https://microbe-notes.come/southern-blot-principle-steps-andapplication/.

34. Blotting methods and application [Internet]. Available form: https://www.web-books.com/Mobio/free/chgD.htm.

35. Denaturing gradient gel electrophoresis [Internet]. Available form:http://wiki.biomine. Skelleffea.Se/wiki/mdex.php/DDGE.

36. Muyzar G., Smalla K. Application of denaturing by gradient gel electrophoresis in microbioecology. AntonieJ.1998;73:127-141.

37. Diez Beatriz, Terence Marsh, Raman Massana. Application of DDGE to study the diversity of marine picoeukaryotic assemble age and comparison of DDGE with other molecular techniques. App and Envr Microbio.2001;67: 2942-2951.

38. Siqueria Jose, Isabela Rocas. Application DDGE to the analysis of Endotoxins infection. J of Endobios.2005; 31: 775-82.

39. Hernango Man S, Espinosa JC. Characterization of nine yeast by TGGE, FEMS Macrobio Let,2000;193:45-50.

40. Harmeet peters, Robbinson Peter. TGGE, Mole Diagno.2005; 32:718-720.

41. Melissa A., Fouratt Nancy, G Love, Ann M. Application of temperature gradient gel electrophoresis to the characterization of anitrifying bioaugmentation product, MicrobioEco.2003;43(2):277-286.

42. Bethan M Jones, Leslie A Knapp. Temporal TGGE for detection of single nucleotide polymorphism. Springer,2009;578:153-165.

43. Hames David, Hopper Nigel. Biochemistry. 3rded. NewYork:Taylor and Francis;2005.

44. BaileyW R, Scott E G, Boron E J. Diagnostic microbiology.St.Louis:Mosby;1986.

45. Sastry AS, Bhat SK. Essential of Medical Microbiology. New Delhi:Jaypee Brother Medical Publisher;2016.

46. Real time PCR [Internet]. available form:https://microbnotes.com/realtime-pcr-principle-process-markers-advantages-applications/.

47. Real time PCR [Internet]. Available form:https://en.m.wikipedia.org/wiki/RPCR.

48. Manisa Z. Wony and Juan F. Medrano. Real time PCR form RNA quantification. Biotech.2018;39(1):207-10.

49. DNA fingerprinting principle and technologies [Internet]. Available form:https://www.yourarticlelibrary.com/dna/dnafingerprinting-priciple and technology of dna-fingerprinting/1221/.

50. Gizaw S, Van Arendonk, JAM Komen. Population structure genetic variations and morphological diversity in indigenous sheep of Ethiopia. Animal Gen.2007; 38:621-628.

51. DNA fingerprinting principle, methods, application [Internet]. Available form:https://microbnotes.com./dna-fingerprinting.-principle-methods-app lication/

52. DNA fingerprinting [Internet]. Available form: https://www.biology exams 4u . com/2014/05/DNA fingerprinting.

53. Koerun Viktor. Molecular markers and their application in cereals breeding.Mol Bio.2015; 12:12-28.

54. Mahesh waran M. Molecular markers. New Delhi: IP Innovative; 2010.

55. Choe Lella, Dutt Michael J., Kelvin H. Studies of potential cerebrospinal fluid molecular markers for Alzheimer's disease. Electropho J.2012;23(14):2247-2251.

56. Damjan Glarae, Michael Dean. Application of heteroduplex analysis for mutation detection in disease genes. Human mutation.1995; 6(4) :201-210.

57. J. Wallae, Andrews R. PCR mutation detection protocol. New Jersey: Human Press;2002.

58. F. El-Yazbi Amira, Wong Aiysha. A luminescent probe of mismatched DNA hybridization. Analyti Chem Acta. 2017; 994:92-99.

59. Tahira T., Kukita Y, Higasa K, Okazaki Y. Estimation of SNP allele frequencies by SSCP analysis of Pooled DNA. Methods in Mol Bio. 2009; 578:193-207.

60. Michael Oto, Satoshi Miyake. Optimization of non radioisotope SSCP analysis with a conventional Gel electrophoresis apparatus. Anal Biochem .1993; 293:19-22.

61. Kubo Karen, Stuart RM, Astua Freitas. Virus by SSCP analysis of nucleotide sequencing of a fragment from the nucleocaprid gene. Arch of Virol .2009;154(6): 1009-14.

62. Masato Orita, Hiroyuki Lawhana. Detection of Ploymorphism of human DNA by Gel electrophoresis as SSCP Process.Nat Acta Sci.1989;86(8) :2766-2770.

63. DNA sequencing [Internet]. Available form: https:// genetic education. come. in/dna-sequencing-hostory-steps-meyhods-application-limitations/.

64. DNA sequencing [Internet]. Available form: https://en.m.wikipedia.org/wikidna sequencing.

65. Lilian Franca, Tarso Kot. A review of DNA sequencing method. Review of Biophys. 2002; 35(2):169-200.

66. Leharach Hans. DNA sequencing method in Human genetics and disease research. Prine Nat.2008; 28:2010-18.

67. Sanger sequencing: Introduction, Principle and protocol [Internet]. Available form: https:// www.cd.geomics.com/blog/sanger sequencing-intro-principle-protocol/.

Received on 06.08.2022 Modified on 27.08.2022

Asian J. Pharm. Ana. 2022; 12(4):271-280.

DOI: 10.52711/2231-5675.2022.00045