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Nucleic acid hybridisation and Nucleic acid probes - Essay Example

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The unique property of nucleic acids to pair with each other through complementary base pairing is the lifeblood of genetic engineering. A single strand of DNA can pair up with another strand of DNA or RNA if its base pairs are…
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Nucleic acid hybridisation and Nucleic acid probes
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The Utility of Nucleic Acid Hybridisation and Nucleic Acid Probes in Molecular Biology by Your Date
The Utility of Nucleic Acid Hybridisation and Nucleic Acid Probes
In Molecular Biology
The unique property of nucleic acids to pair with each other through complementary base pairing is the lifeblood of genetic engineering. A single strand of DNA can pair up with another strand of DNA or RNA if its base pairs are complementary to those of the other strand, under the right conditions of temperature and pH. This phenomenon is called nucleic acid hybridisation. It is possible to exploit this mechanism for the detection of one nucleic acid strand from a mixture of many other strands. For instance, if a DNA strand with a desired nucleotide sequence is to be detected from a mixture of many other strands, an oligonucleotide containing a few complementary bases to the desired sequence can be prepared and attached to an anchor such as a membrane or a paper. When soaked in a solution having a mixture of many strands, the one, which is complementary to the oligonucleotide, will bind to it through complementary base pairing, also known as “zippering” (Lodish et al, 2004, p. 11).
When double stranded DNA is heated in a dilute salt solution, its two strands separate because of the breakdown of complementary base pairing (melting). This strand separation is called denaturation. The temperature at which the two complementary strands separate is called the melting temperature ‘Tm’, and is affected by the percentage of G.C base pairs, ion concentration of the solution, presence of destabilising compounds like urea, and the pH of the solution (Lodish et al, 2004, p. 105).
Nucleic acid probes
A particular fragment of DNA or RNA whose nucleotide sequence is complementary to a gene or nucleotide of interest is called a nucleic acid probe. A nucleic acid probe has to be designed in such a way that it hybridises through complementary base pairing to the target DNA or RNA that has to be detected. It should be long enough (about 20 nucleotide long) to pair exclusively to the target nucleotide sequence. Probes are labeled with radioactive tracers, histochemical compounds or fluorescent dyes to enable their detection from a heterogeneous mixture of nucleic acids (Nussbaum et al, 2004, p.41). For instance, 32P labeled probes are developed using polynucleotide kinase that transfers a 32P labeled phosphate group from ATP to the 5’ end of the probe (Lodish et al, 2004, p. 369). Autoradiography by exposure to an x-ray film can be used for detection of radiolabelled probes while fluorescent microscopes, fluorescence scanners, spectrofluorometers and microplate readers can be used for the detection of fluorescent labeled probes (“Fluorescent Probes”). Examples include Fluorescent insitu hybridisation (FISH) and DNA microarrays where probes are used to detect specific target sequences.
Nucleic acid analysis through hybridisation
Various tests in molecular biology depend on the use of nucleic acid hybridisation. Apart from PCR, nucleic acid hybridisation is employed in Southern hybridisation, Northern hybridisation, Dot blot, etc.
Southern blot: It is named after Dr. Edward M. Southern who developed this procedure at Edinburgh University. This technique is used to detect a particular DNA fragment. Firstly, the DNA is cut using restriction endonucleases. The DNA fragments thus cleaved are separated using agarose gel electrophoresis based on their size, followed by alkaline denaturation. The separated DNA is then transferred onto a nitrocellulose membrane (blotting). The membrane is placed on a gel that is placed in a buffer. An absorbent paper or a sponge is placed below the gel that enables the buffer to be drawn up through it, thereby transferring the DNA onto the membrane. The membrane is then placed in a solution containing labelled probes that will hybridise to the target DNA, which can then be detected using a suitable technique based on the kind of probe used.
Northern blot: This technique is similar to Southern blot. The only difference is that while Southern blot is used to detect a specific DNA sequence, northern blot is used to detect and RNA sequence.
These two techniques are widely used for screening of genomic libraries, c-DNA libraries, determination of the number of copies of a particular gene, detecting gene defects etc.
Dot and slot blot: These techniques are used for analyzing human genomic DNA, which is blotted onto a nitrocellulose membrane, followed by denaturation by heat or alkali. They are then subjected to a probe. These techniques can be used to distinguish between alleles that vary even by single nucleotides (“Nucleic acid hybridization assays”, 1999, Ch. 5).
Nucleic acid hybridisation is used in many routine experiments in the molecular biology laboratory, making it an indispensable requirement in genetic engineering and molecular biology.
References
“Fluorescent Probes”, n.d. piercenet.com. Retrieved August 2, 2011 from http://www.piercenet.com/browse.cfm?fldID=4DD9D52E-5056-8A76-4E6E-E217FAD0D86B
Lodish, H. et al., 2004. Molecular Cell Biology. W. H. Freeman, New York.
“Nucleic acid hybridization assays”, 1999. Retrieved August 2, 2011 from http://www.ncbi.nlm.nih.gov/books/NBK7567/
Nussbaum, R. L., 2004. Thompson and Thompson genetics in medicine. Elsevier Health Sciences, n.a. Read More
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