molecular biology techniques

molecular biology techniques

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  MOLECULAR BIOLOGY TECHNIQUES •Molecular biologyis the branch of biology that deals with the molecular basis of biolgical activity. •This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. •It concerns itself with understanding and the interactions between various systems of cells like DNA,RNA and protein biosynthesis.
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  Relationship to otherbiological sciences
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  • Biochemistry: Isthe study of chemical substances and vital processes occuring in living organisms. • The study of chemistry behind biological processes and the synthesis of biologically active molecules are examples of biochemistry. • Genetics: Is the study of effect of genetic differences on organisms. • The study of “mutants” organisms which lack one or more functional components with respect to the so called “wild type” or normal phenotype. • Molecular biology: Is the study of molecular process of replication,transcrption,translation and cell funcion. • The central dogma of molecular biology where genetic material is trsnscribed into RNA and then translated in to proteins.
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  TECHNIQUES OF MOLECULAR BIOLOGY 1.Expression cloning 2. Polymerase chain reaction(PCR) 3. Gel electrophoresis 4. Macromolecule blotting and probing techniques • Southern blotting • Northern blotting • Western blotting • Eastern blotting 5. Arrays 6. Allele specific oligonucleotide 7. Antiquated technologies
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  • Expression cloning:One of the most basic techniques of molecular biology to study protein function is expression cloning. • In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). • This plasmid can be inserted into either bacterial or animal cells. • Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). • Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. • The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection. • In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. • The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.
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  • Polymerase chainreaction (PCR):It is an extremely versatile technique for copying DNA. • In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. • For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". • PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.
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  • Gel electrophoresis: •The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. • In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. • Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.
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  • Southern blotting: •Named after it’s inventor,biologist Edward southern • It’s a method of probing for prescence of specfic DNA sequence with DNA sample • DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. • The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. • Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples
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  • Northern blotting: •The northern blot is used to study the expression patterns of a specific type of RNA molecule as relative comparison among a set of different samples of RNA. • In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. • The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. • The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. • It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.
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  • Western blotting: •In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). • The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. • This membrane can then be probed with solutions of antibodies. • Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. • Using western blotting techniques allows not only detection but also quantitative analysis.
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  • Eastern blotting: •Eastern blotting technique is to detect post- translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates. • Further combinations of these techniques produced such terms as southwesterns(protein-DNA hybridizations), northwesterns (to detect protein-RNA interactions) and farwesterns (protein-protein interactions), all of which are presently found in the literature.
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  • Arrays: • ADNA array is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragment. • Each spot has a DNA fragment molecule that is complementary to a single DNA sequence (similar to Southern blotting). • A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling). • In this technique the RNA in a tissue is isolated and converted to labeled cDNA. This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done. • Since multiple arrays can be made with exactly the same position of fragments they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. • Also, one can measure what genes are expressed and how that expression changes with time or with other factors. • Arrays can also be made with molecules other than DNA. For example, an antibody array can be used to determine what proteins or bacteria are present in a blood sample.
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  • Allele SpecificOligonucleotide: • It is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. • Short (20-25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA. • Hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. • The target DNA is then washed and the labeled probes that didn't hybridize are removed. • The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation. • The Illumina Methylation Assay is an example of a method that takes advantage of the ASO technique to measure one base pair differences in sequence.
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  • Antiquated technologies: •In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. • For example, before the advent of DNA gel electrophoresis (agarose or polyacrylamide), the size of DNA molecules was typically determined by rate sedimentation in sucrose gradients, a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used. • Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate.
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