Molecular Probes and Their Applications

Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

K Vasavirama1*

*Corresponding Author: K Vasavirama,  [email protected]

Molecular probes are small DNA or RNA segments that recognize complementary sequences in DNA or RNA molecules that allow identification and isolation of these specific sequences from an organism. Extraordinary advances in human molecular genetics have occurred over the past decade which has revolutionized the entire scenario of biological sciences. One component of those advances is the increase in the use of molecular probes. These probes serve as the resources for a variety of applications. These molecular markers have acted as versatile tools and have found their own position in various fields like taxonomy, physiology, embryology and genetic engineering.

Keywords: DNA probe, RNA probe, cDNA probe, RFLP, DNA fingerprinting

INTRODUCTION Although, initially these probes were developed and used for genetic engineering research but A stretch of DNA or RNA sequence that can detect a target sequence in the genome is called as are now frequently used for a variety of purposes probe. Ever since their development, they are including diagnosis of infectious diseases constantly being modified to enhance their utility (Katoch et al., 1994,1997; Kaminski et al., 1995; and to bring about automation in the process of Sharma et al., 1996; Belak et al., 2009;Bexfield genome analysis. The discovery of PCR (Mullis et al., 2011; Palacios et al., 2009), identification et al., 1986; Saiki et al., 1988) was a landmark in of food contaminants (Zhang et al., 2012; Goji et this effort and proved to be a unique process that al., 2012), variety of microbiological tests (Chuba brought about a new class of DNA profiling et al., 1998;Smorawinska et al., 1992) and markers. This facilitated the development of forensic tests. Probes can also be used to identify marker-based gene tags, map-based cloning of different varieties of crop species (Cordeiro et al., agronomically important genes, variability studies, 2001; Anderson et al., 1993; Nagaraju et al., phylogenetic analysis, marker-assisted selection 2002). For basic studies in molecular biology of desirable genotypes etc. laboratories these are frequently used for

1 Department of Biotechnology, Gitam Institute of technology, Gitam University, Visakhapatnam – 530045.

This article can be downloaded from http://www.ijlbpr.com/currentissue.php 32 Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013 identification and isolation of genes or related fragments of different sizes. Isolate DNA of sequences. specific fragment from a particular band identified through southern blots by hybridization with In theory any nucleic acid can be used as a specific labeled mRNA or cDNA molecules. Clone probe provided it can be labeled to permit this DNA in a vector. Allow chimeric vector to infect identification and quantitation of the hybrid bacteria for multiplication where it can make molecules formed between the probe and billions of copies. DNA probes prepared in this sequence to be identified. In practice, double and manner can be used for southern blotting and single standard DNAs, mRNAs, and other RNAs RFLP analysis. synthesized in vitro are all used as probes. DNA/ RNA probe assays are faster and sensitive so RNA Probes that many conventional diagnostic tests for High specific activity RNA probes or riboprobes viruses and bacteria involving culturing of the may also be synthesized from DNA templates organisms are being fast replaced by molecular cloned in expression vectors such as SP6 (which probe assays. While culture tests can take days infects Salmonella typhimurium) and T7 phage or even months, molecular probe assays can be (infects E.coli). This is achieved through RNA performed with in few hours or minutes. synthesized in vitro and labeled simultaneously Molecular probes can be broadly categorized with labeled nucleotides. into DNA probes and RNA probes, sometimes Usually SP6 and T7 systems can be and have cDNA probes and synthetic oligonucleotide been utilized to express whole RNAs, but for probes can also be used for various purposes. making a probe, only a short labeled RNA is The use of molecular probes has become sufficient. To enable such probes to be transcribed todays most sophisticated and sensitive into uniform lengths, it is practice to linearize the technology for a variety of uses involving plasmid by cleaving it with a restriction enzyme. biological systems both in basic and applied The vector thus carries the input in the following studies in the field of molecular biology and order phage promoter-Enzyme 1- Enzyme 2. The biotechnology including their commercial use. template DNA is inserted at the number 1 site and composite is treated with restriction enzyme Preparation of Probes 2. So now this creates a linear DNA with a Different types of probes can be prepared in promoter, the template DNA and automatic various ways. termination site at the end cleaved with enzyme DNA Probes 2. The mRNAs fall of when they reach this end of Extract the DNA from an animal or plant the vector. Such templates reflect to run of tissue.Digest extracted DNA with a restriction templates and are uniform in size and are easier enzyme such as EcoRI or Hind III which cuts DNA to isolate from the reaction mixture. RNA probes at specific sites or positions where a specific prepared in this manner can be used for northern sequences recognized by the enzyme is found. blotting and in situ hybridization. Run the digested DNA on an agarose or RNA probes offer several advantages over polyacrylmide gel electrophoresis to separate DNA probes. Since these are single stranded and

This article can be downloaded from http://www.ijlbpr.com/currentissue.php 33 Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013 provide improved signal or hybridization blots. End labeling There is lack of competition of probe/ probe In this technique probe is isolated and end labeled hybridization. Even though, some advantages are by removing the 5'-terminal phosphate using there wide spread presence of ribonuclease alkaline phosphatase first and adding a creates some problems in their preparation and 32P-labeled phosphate with the help of a kinase. use. So RNA probes are more sensitive to End labeled probes are far less labeled than the degradation than equivalent DNA probes, transcribed ones with labels at several therefore extreme care must be taken in the nucleotides in the strand. preparation of RNA probes by keeping all glass Nick Translation ware free of ribonuclease. It is one of the commonly used techniques for cDNA Probes producing a radioactive probe. A purified phage A DNA sequence corresponding to a part of a or plasmid vector containing a cloned genomic specific gene can be obtained by reverse or cDNA sequence is treated with a small amount transcription of mRNA. cDNA thus obtained can of pancreatic DNase which hydrolyzes the be cloned and used as a probe. phosphodiester bonds between nucleotides. At Synthetic oligonucleotides as probes very low concentration the DNase produces only scattered “nicks” in one or other strand of the DNA probes with known nucleotide sequence can duplex DNA. DNA polymerase and radioactively also be synthesized chemically using automated labeled deoxynucleotides are also added to the DNA synthesizers. These synthetic probes will DNA sample. Using the unharmed strand as be efficient only when they are not more than 20- template, the DNA polymerase synthesizes a new 40 nucleotides in length. second strand using exposed 3' end at a nick Labeling of probes site as primer, which then displaces the existing The detection of homologous sequences after DNA from the 5’ end of the nick. Radioactive hybridization with the probe is like finding a needle nucleotides are incorporated into the new strand, in the hay-stock. Therefore, for the success of so, a single standard probe is created when the DNA probe assay it is necessary to develop duplex DNA is denatured. simple, safe and sensitive techniques for their use. As probes transmit no signal of their own Choice of Label they have to be either labeled with radioactive Probes can be labeled either by radioactive isotopes or coupling of non-radioactive signal isotopes or can also be labeled with non- molecules to the probes without impairing the radioactive molecules such as biotin, digoxegenin hybridization ability of these probes. These signal etc. molecules may include fluorescent antibodies, Radiolabeled Probes enzymes that produce color changes in dyes and The most sensitive detection method employed chemiluminescent catalysts. is radioactive label. Traditionally radioactively Methods for Labeling of Probes labeled probes are used for a variety of There are two methods for labeling of probes. experiments. The autoradiographic detection of i.e. end labeling and nick translation. labeled probes depends on the isotope used and

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the specific activity which has to be high enough Table 2: Merits of Radiolabeled and to permit detection after hybridization within a Non-radiolabeled Probes reasonable exposure of time or with a good Label Resolution Sensitivity Exposure Stability signal. Several isotopes are available for 32 p + ++ 7 days 0.5 weeks radioactive labeling. The use of 32P allows rapid detection of signal yet cellular localization is 35 S ++ +++ 10 days 6 weeks 32 suboptimal because of the long path of the [ P] 3H +++ +++ 14 days >30 weeks -rays. Autoradiographs of high quality and Biotin +++ ++ 0.16 days >52 weeks improved cellular localization employ 35S labeled probes. 35S emits  -rays of much shorter path Digoxigenin +++ ++ 0.16 days >52 weeks length. One of the major disadvantages of 35 S is and 2. non-specific binding of label to cell or tissues. It has been suggested that pre-hybridization in the Non-Radiolabeled Probes presence of non-labeled thio alpha UTP at pH 5.5 a) Biotin Labeled Probes reduces this non-specific binding. It is also Recent advances in nucleic acid technology now possible to label nucleic acids with tritium or by offer alternatives to radio activity labeled probes. iodonization with [125 I] iodine. Tritium labeled One of such procedure that is becoming popular probes give intracellular localization, but for low abundance target molecules may require is biotin labeling of nucleic acids. This system exposure of up to 100 days. This may result in exploits the affinity which the glycoprotein avidin high background and is unacceptably long for has for biotin. Avidin is commonly found in egg most purposes. Disadvantage with radiolabel is white. Biotinylated probes are prepared through Instability of label. Different labels along with the a nick-translation reaction by replacing merits and demerits were presented in Tables 1 nucleotides with biotinylated derivatives. After hybridization and washing, detection of hybrids Table 1: Different Types of Labels for Labeling of Probes is done by a series of cytochemical reactions which finally give a blue color whose intensity is Radiolabeled Probes Non-radioactive Labelled Probes proportional to the amount of biotin in the hybrid. 125 3H, 32P, 35S,14C, 1 Digoxigenin Advantages 4-(Phenoxymethyl)piperidine Biotine These assays employ non-toxic materials, whose 5-Bromo deoxyuridine half-life is longer. Dinitro phenyl • These probes can be prepared in advance in Alkaline Phosphate bulk and stored at -20°C for repeated uses. Ethidium • Detection of hybrids is much faster than by Sulphonation radioactive probes. Luciferase

Sodium metabisulphate LIMITATIONS

Mercury Meta Acetate • A limitation of this technology is that very small probe contains only a small number of TetramethylRhodamine

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biotinylated sites limiting the intensity of signal been devised for attaching other ligands (eg: obtained. It has been solved by adding long hapten determinants, 2, 4 dinitrophenol, ‘tails’ of biotinylated nucleotides to the probes arsenative derivatives etc) to nucleotides without through enzymatic methods. Sometimes the hampering their ability to be incorporated into probe does not need to be labeled with biotin DNA. These alternatives require binding of but only coupled with a tail. attached ligands to specific proteins that can be tagged with enzymes or fluorescent molecules. • Another disadvantage of biotin labeled probes It is possible to monitor many probes is that cytochemical visualization reactions simultaneously by using several different ligands lead to precipitation of insoluble material which since each ligand would yield a different signal. A cannot be removed and therefore the filter chemiluminscent probe system has also been cannot be reused, whereas with radio labeled developed in which two different probes probes, the filters can be used repeatedly for complementary to a continuous segment of DNA hybridization with a number of probes one at hybridize to adjacent segments of a gene. The a time. first label is a chemiluminescent complex that b) Digoxigenin Labeled Probes emits light at a specific wavelength this emission Digoxigenin is another chemical derived from excites the label molecule on the second probe plants and used for non-radioactive labeling of to emit light at a different wave length which can probes. An antibody associated with an enzyme be detected using a photomultiplier device: This (antidigoxigenin - alkaline phosphatase conjugate) process called non-radioactive energy transfer is used for the detection of the presence of can occur only if the two probes hybridize digoxigenin. The probes may be labeled with correctly and the two labels are close to each digoxigenin - II - dUTP supplied with a digoxigenin other. This system has great fidelity and provides kit (these kits are available from any commercial basic technology for a homogeneous assay. In firm, eg. BoehringerMannheem). The labeled and addition DNA does not need to be immobilized denatured probe may be used for hybridization. and no washing steps are necessary which may After hybridization the membrane or the slide may be an additional advantage for large scale testing. be transferred into detection buffer containing 20 ug/ ml of antidigoxigenin fluorescein and 5% Applications of Molecular Probes (W/V) BSA (bonine serum albumin). This is Molecular probes are used in restriction fragment incubated for 1 h at 37°C and then the membrane length polymorphisms (RFLPs) and related or slide is washed in detection buffer three times Analysis (8 min each at 37°C) and alkaline phosphatase a) RFLPs for Evolutionary Studies activity was detected using 0.17 mg/ml BCIP (5- The restriction fragment length polymorphisms bromo 4-chloro 3-indoyl phosphate) and 0.33 mg/ (RFLPs) can be studied in a set of related ml NBL (nitro blue tetrazolium) as dye substrate. species using a random or a specific DNA probe. c) Alternatives to Biotin and Digoxigenin The similarities and differences can be used to Labeling infer phylogenetic relationships. This has actually The techniques of non-radio isotopic labeling have been done in a number of cases both in plants been further expanded and new methods have

This article can be downloaded from http://www.ijlbpr.com/currentissue.php 36 Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013 and in animals(Gill et al., 1991; Fukuchi et al., identified through RFLP mapping analysis which 1993; Moore et al., 1991; Mason-Gamer demonstrates power of RFLP/Linkage analysis. et al.,1998; Deshpande et al., 1998; Drinkwater Use of Molecular Probes in Molecular et al., 1991; Mburu and Hanotte, 2005; Guimaraes, Cytogenetics 2007) a) Isolation of Genes Using DNA or RNA b) RFLP Maps and Linkage of RFLPs with Probes Specific Genes Specific molecular probes can be used for RFLPs have been used to prepare chromosome isolation of specific genes. These probes may maps in humans, mice, fruit fly and in plants be available either from same species or from including maize, tomato, lettuce, and rice. The another species can be used for isolation of use of Mendelian markers for genetic mapping is genes. If probes obtained from one species used sometimes limited due to non-availability of for isolation of gene from the same species they mutants. The list of markers can be increased or are called as homologous probes. If probes extended by using molecular markers which are obtained from another species used for isolation examined in the form of RFLPs. Once a large of genes in other species they are defined as number of RFLPs are available in a species the heterologous probes. parents, F , F generations can be used to study 1 2 These heterologous probes have been found their inheritance and linkage relationship and to be effective in identifying gene clones during genetic linkage maps can be prepared. In this way colony hybridization or plaque hybridization or on they can be used for plant and animal breeding southern blots. For instance, the gene for (Mburu and Hanotte et al., 2005; Guimaraes et chalcone-synthase (CHS) has been isolated from al., 2007; Parasnis et al., 1999; Sen et al., 1997; Antirrhinum majus and Petunia hybrida using Pujar et al., 1999; Sant et al., 1999) heterologous cDNA probes from parsley similarly c) RFLP Markers to Map the Genes in heterologous Antirrhinum cDNA probe was used Diseased Persons and Identification of for isolation of CHS gene from barley and Disease heterologous probes from maize were used for DNA polymorphisms (many forms) are isolation of barley genes Wx (Waxy Genes) and differences in DNA sequence that result from Al (aleurone gene). Heterologous probes should point mutations, random deletions or insertions ordinarily be used with cDNA library and not with or the presence of varying number of repeated the genomic library since in the latter case copies of a DNA fragments (tandem repeats). A unrelated genes or pseudogenes (which do not polymorphism in the coding region of a gene may express) may be isolated and cloned. These be detected as an alteration in the amino acid heterologous clones if available in expression sequence of the encoded protein. It is now vector [Eg. PGEM Blue] can also be used for possible to detect polymorphisms in unexpressed getting RNA probes which have been bound to regions of DNA by this analysis. Genetic be more sensitive and efficient. disorders like sickle cell anemia, Thalassemia’s, b) In situ Hybridization Huntington’s disease and cystic fibrosis were

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In situ hybridization (ISH) is a technique which As represented in Table 3 the DNA probes are permits detection of DNA or RNA sequences in being extensively utilized for diagnosis of cell smears, tissue sections and metaphase diseases caused by parasitic protozoa and chromosome spreads. The method is based on helminthes. They are also used for antenatal the formation of double stranded hybrid molecules diagnosis of congenital diseases to allow advice which form between a DNA or RNA target on abortion of fetus, if desired. Similarly probes sequence and the complementary single have been designed for the diagnosis of number standard labeled probe. In a number of cases rye of sexually transmitted diseases. chromosomes in wheat background have been Readymade DNA probes for herpes virus and identified using this technique. other human, animal plant viruses are available. Satellite chromosomes with NORs (nuclear Probes are also available for a number of human organizing regions) are recognized using probes parasites from the groups protozoa and for ribosomal DNA in wheat, barley etc. Telomeres helminthes. In India, a diagnostic probe for the have been identified in human and other detection of malaria has been constructed at the eukaryotes using a telomeric sequence as a Astra Research Centre India (ARCI). molecular probe showing that telomeres of all chromosomes carry the same sequence. In USE OF MOLECULAR PROBES wheat and related species chromosomes of D IN DNA FINGER PRINTING genome can be identified using a D genome DNA fingerprinting is a way of identifying a specific specific probe (PASI separated from Aegilops individual, rather than simply identifying a species squarrosa). or some particular trait. It is also known as genetic This method is particularly useful if target fingerprinting or DNA profiling. Inventor of this sequences are distributed in a non-random way technology is Sir Alec Jeffreys in 1985. DNA in tissues for the visualization of heterogenicity fingerprinting is currently used both for identifying and the study of cell differentiation. The method paternity or maternity and for identifying criminals of ISH for RNA-RNA hybridization is generally or victims. applicable to rapid screening of small numbers The vast majority of a human’s DNA will match of cultured cells for expression of oncogenic exactly that of any other human, making mRNA. It circumvents the problem of extracting distinguishing between two people rather difficult. rare RNAs in sufficient amounts for detection. DNA fingerprinting uses a specific type of DNA Similar methods have been used to visualize sequence known as Variable Number Tandem homeotic gene expression in developing larvae. Repeats (VNTRs) can contain anywhere from DNA-DNA ISH is also suitable for the detection of twenty to one hundred base pairs. Every human viral genomes in sections of routinely processed being has some VNTRs. To determine if a person archival paraffin blocks of human tissues. has a particular VNTR, a southern Blot is USE OF MOLECULAR PROBES performed and then the southern blot is probed FOR HUMAN HEALTH CARE through a hybridization reaction with a radioactive

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Table 3: DNA Probes for Different Diagnostic Tests

Parasite Probe Used HybridizationTechnique Used

I Protozoa

1. Leishmania Kinetoplast DNA (KDNA, a repetitive DNA Dot blots, southern blots In situ hybridization (Kala-azar) sequence)

2. Trypanosoma Kinetoplast DNA (KDNA) a repetitive DNA Southern Blot (Restriction Fragment (Sleeping sickness) and total parasite DNA Length Polymorphism RFLP

3. Plasmodium Repetitive DNA, Synthetic oligonucleotides Dot blots, blood-samples lysed directly on (Malarian Parasite; nitrocellulose filters P. falciparum P. vivax and P. cynomorge)

II Helminths

1. Schistosomes Ribosomal RNA gene Dot blots, Southern blots (human blood flukes)

2. Nematodes: Repetitive DNA sequences; Synthetic Crude DNA Blood Samples dot blots Wuchereria and oligonucleotids; Ribosomal DNA; Brugia (filaria) onchocerca (river blindness); Trichinella

3. Taeniid Cestodes Ribosomal DNA Southern blots (Taenia solium) version of the VNTR in question. The pattern of high molecular weight DNA, complete digestion which results from this process is what is often of the samples with appropriate enzymes and referred to as a DNA fingerprint. perfect transfer and hybridization of the blot to obtain distinct band with appropriate control. If DNA fingerprinting has a high success rate and necessary the analysis should be repeated. The a very low false-positive rate making it an DNA fingerprinting test is performed properly is extremely popular form of paternity and maternity infallible. However, there is need to interpret DNA verification. In forensics DNA fingerprinting is very typing with a thorough understanding of the attractive because it requires only a piece of a population involved. person’s body like blood, hair, saliva, semen, body tissue cells. DNA finger printing can be used for: DNA finger printing is technically demanding. 1) Pedigree analysis and establishing paternity. There are cases on record in the US where 2) Immigration authorities. mistakes seem to have been prompted by human 3) In rape cases. error. There is a need for quality control. Every precaution should be taken to ensure preparation 4) Identification of mutilated dead bodies.

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5) Social security record identification. 2. Belak S, Thoren P, Leblanc N and Viljoen G (2009), “Advances in Viral Disease 6) In murder cases. Diagnostic And Molecular Epidemiological 7) In characterization of cell cultures. Techniques”, Expert Rev. Mol. Diagn., Vol. 8) In animal breeding programmes. 9, No. 4, pp. 367-381. 9) In plant breeding programmes. 3. Bexfield N and Kellam P (2011), “Metagenomics and the Molecular 10) In demographic studies. Identification of Novel Viruses”, Vet. J., Vol. 11) Twin zygosity determination. 190, pp. 191-198. 12) Tissue culture cell line identification. 4. Chuba P J, Pelz K, Krekeler G, de Isele T S 13) Mopping fine grained sequence variation in and Gobel U (1998), “Synthetic mini satellites. Oligodeoxynucleotide Probes For The Rapid Detection Of Bacteria Associated CONCLUSION With Human Periodontitis”, J Gen Since Molecular probes assays are most Microbiol., Vol. 134, pp. 1931-1938. sensitive than conventional diagnostic methods, 5. Cordeiro G M, Casu R, McIntyre C L, their use has become today‘s most sophisticated Manners J M and Henry R J (2001), and sensitive technology for a variety of uses “Microsatellite Markers from Sugarcane involving biological systems both in basic and (Sacharum spp.) ESTs Cross Transferable applied studies. They give new dimensions to to Erianthus and Sorghum”, Plant Sci., Vol. concerted efforts of breeding and marker-aided 160, pp. 115-1123. selection that can reduce the time span of 6. Deshpande A D, Ramakrishna W, Mulay G developing new and better varieties and will make P, Gupta V S and Ranjekar P K (1998), the dream of super varieties come true. This ideal “Evolutionary and Polymorphic Organization technology would offer absolute specificity, of the Knotted-1 homeobox in Cereals”, modularity, minimal size, deliverability (access to Theor Appl Genet., Vol. 97, pp. 135-140. all cell types) and physiological neutrality (non- cytotoxic, biochemically). In this way, their use 7. Drinkwater R D and Hetzel D J S (1991), has become most versatile in the field of “Application of Molecular Biology to molecular biology and biotechnology including Understanding Genotype-environment their commercial use. Interactions in Livestock Production”, In Proc. of an International Symposium on REFERENCES Nuclear Techniques in Animal Production 1. Anderson J A, Sorrells M E and Tanksley S and Health, IAEA, FAO, Vienna, April 15- D (1993), “RFLP Analysis of Genomic 19, pp. 437-452. Regions Associated With Resistance to 8. Fukuchi A, Kikuchi F and Hirochika H (1993), Preharvest Sprouting in Wheat”, Crop Sci., “DNA fIngerprinting of Cultivated Rice With Vol. 33, pp. 453-459. Rice Retrotransposon Probes”, Jpn. J.

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Genet., Vol. 68, pp. 195-204. A (1998), “Granule-Bound Starch Synthase: 9. Gill K S, Lubbers E L, Gill B S, Raupp W J, Structure, Function, and Phylogenetic and Cox T S (1991), “A Genetic Linkage Map Utility”, Mol Biol Evol., Vol. 15, No. 12, pp. of Triticumtauschii (DD) and its Relationship 1658-1673. to the D Genome of Bread Wheat 16. Moore S S, Sargeant L L, King T J, Mattick (AABBDD)”, Genome, Vol. 34, pp. 362-374. J S, Georges M and Hetzel D J S (1991), “The Conservation Of Dinucleotide 10. Katoch V M and Sharma V D (1997), Microsatellites Among Mammalian “Advances in the Diagnosis of Mycobacterial Genomes Allows the Use of Heterologous Diseases”, Indian J Med Microbiol., Vol. 15, PCR Primer Pairs in Closely Related pp. 49-55. Species”, Genomics, Vol. 10, pp. 654-660. 11. Katoch V M, Kanaujia G V, Shivannavar C T 17. Mullis K, Faloona F, Scharf S etal. (1992), et al. (1994), “Progress in Developing “Specific Enzymatic Amplification of DNA In Ribosomal Rna And Rrna Gene(s) Based Vitro: The Polymerase Chain Reaction”, Probes For Diagnosis And Epidemiology of Biotechnology, Vol. 24, pp. 17-27. Infectious Diseases Especially Leprosy”, in Sushil Kumar, Sen A K, Dutta G P Sharma 18. Nagaraju J, Kathirvel M, Kumar R R, Siddiq R N (Eds.), Tropical Diseases - Molecular EA and Hasnain S E (2002), “Genetic Biology and Control Strategies, pp. 581- Analysis of Traditional And Evolved Basmati 87A, CSIR Publication, New Delh. and Non-basmati Rice Varieties by Using Fluorescence Based ISSR-PCR and SSR 12. Kaminski D A and Hardy D S (1995), Markers”, Proceedings of the National Selective Utilization of DNA Probes for Academy of Sciences, USA, Vol. 99, pp. Identification of Mycobacterium species on 5836-5841. the Basis of Cord Formation in Primary BACTEC Cultures”, J ClinMicrobiol, Vol. 33, 19. Noriko Goji, Trevor MacMillan and Kingsley pp. 1548-50. Kwaku Amoako (2012), “A New Generation Microarray for the Simultaneous Detection 13. Koebner R M D, Powell W and Donini and Identification of Yersinia pestis and (2001), Contributions of DNA Molecular Bacillusanthracis in Food”, Journal of Marker Technologies to the Genetics and Pathogens, Vol. 2012, pp. 1-8. Breeding of Wheat and Barley”, Plant breeding Review, Vol. 21, pp. 181-220. 20. Parasnis A S, Ramakrishna W, Chowdari K V, Gupta V S and Ranjekar P K (1999), 14. McFadden J J, Kunze Z and Seechum P “Microsatellite (GATA)n Reveals Sex-specific (1990), “DNA probes for Detection and Differences in Papaya”, TheorAppl Genet, Identification”, in McFadden J (Ed.), Vol. 99, pp. 1047-1052. Molecular Biology of the Mycobacteria, 21. Palacois G, Druce J, Du L, Tran T, Birch C, Surrey University Press, UK, pp. 139-172. Briese T, Conlan S, Quan P L, Hui J, Marshall 15. Mason-Gamer R J, Weil C F and Kellogg E J, Simons J F, Egholm M, Paddock C D,

This article can be downloaded from http://www.ijlbpr.com/currentissue.php 41 Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013

Shieh W J, Goldsmith C S, Zaki S R, Catton DNA with a Thermostable DNA Polymerase”, M. and Lipkin W I (2008), “A New Arenavirus Science, Vol. 239, pp. 487-491. in a Cluster of Fatal Transplant-Associated 24. Smorawinska M and Kuramitsu H K (1992), Diseases”, New Engl. J. Med., Vol. 358, No. “DNA Probes for Detection of Cariogenic 10, pp. 991-998. Streptococcus Mutans”, Oral 22. Sharma R K, Katoch K, Shivannavar C T et MicrobiolImmunol, Vol. 7, pp. 177-81. al. (1996), “Comparisons of Sensitivity of 25. Zhang X, Wu S, Li K, Shuai J, Dong Q Probes Targeting RNA vs DNA in Leprosy and Fang W (2012), “Peptide Nucleic Acid Cases”, Indian J Med Microbiol.,Vol. 14, pp. Fluorescence in situ Hybridization for 99-104. Identification of Listeria genus, Listeria 23. Saiki R K, Gelfand D H, Stoffel S et al. (1988), monocytogenes and Listeria ivanovii”, Int J Food Microbiol., Vol. 157, No. 2, pp. 309-313. “Primer-Directed Enzymatic Amplification of

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