Molecular Probes and Their Applications
Total Page：16
File Type：pdf, Size：1020Kb
Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013 ISSN 2250-3137 www.ijlbpr.com Vol. 2, No. 2, April 2013 © 2013 IJLBPR. All Rights Reserved Review Article MOLECULAR PROBES AND THEIR APPLICATIONS 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 This article can be downloaded from http://www.ijlbpr.com/currentissue.php 34 Int. J. LifeSc. Bt & Pharm. Res. 2013 K Vasavirama, 2013 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.