Techniques of Molecular Genetics Before we look at specific techniques of molecular genetics, we need to understand the general purposes of these

techniques. There are many practical applications for these techniques, but they all involve one of just a few general purposes. These basic reasons for using molecular techniques are as follows: Amplification Separation and Detection Expression

As each technique is discussed, we will look at how it accomplishes one of these purposes. Formation of Recombinant DNA Most of the techniques of molecular genetics are either directly or indirectly dependent on molecules discovered in and isolated from bacteria. These molecules have functions in the everyday life of bacteria, but scientists have learned to exploit them as tools used for the manipulation and study of DNA. One vital type of molecule discovered in bacteria is the restriction endonuclease (also known as the restriction enzyme). These enzymes were discovered in 1970 (their discoverers won the Nobel Prize in 1986 for their work), and have since been isolated from hundreds of different species and strains of bacteria, meaning there are hundreds of different restriction enzymes. So what do these enzymes do? The name endonuclease tells us their function - nuclease is an enzyme that cuts (or cleaves) nucleic acids (such as DNA in this case), and endo means 'within'; therefore, the enzyme cuts within a DNA molecule rather than at the ends. In bacteria, this activity is used to protect bacteria from invasion by bacteriophages - when the bacteriophage injects its DNA, the enzymes cut the DNA and prevent replication of the phage. At least, that's the idea; the strategy isn't always successful. Restriction enzymes are named according to the species and strain of bacteria they are isolated from. The first letter of the name comes from the genus of the bacterium, the next two letters come from the species, and any subsequent designations are the strain of bacterium and the order in which the enzyme was discovered in that bacterium. For example, the enzyme EcoRI was discovered in Eschericia coli, strain RY13, and it was the first restriction enzyme identified in that bacterium (hence the designation I). These enzymes recognize specific base sequences within DNA molecules, and cleave specific phosphodiester bonds within that sequence. Different enzymes recognize different sequences, but the sequences recognized share one property - they are all palindromes. A palidrome is a word or phrase that reads backwards the same as it does forwards. In English, this can be a phrase like, "Madam, I'm Adam" or "Sit on a potato pan, Otis". In molecular biology, a palindromic base sequence is one that reads the same in one direction on one strand as it does the other direction on the other strand. (And because DNA strands are antiparallel, that means both sequences are read 5' to 3'.) For example, the palindromic sequence recognized by the restriction enzyme EcoRI is GAATTC. Note (as can be seen in the diagram below) that this sequence (highlighted in red) reads GAATTC on the other strand as well.

When EcoRI encounters its recognized sequence, it cuts the phosphodiester bond between the G and the first A on both sequences (indicated by the green arrows in panel B). This causes the two segments of DNA to separate (as shown in panel C). Note that each fragment of DNA has a four base single-stranded 'tail' protruding from one end. Such a single-stranded projection is often called a "sticky end", which will come into play shortly. Restriction enzymes are very powerful and useful tools, because they allow researchers to cut large DNA molecules into smaller, more easily manipulated fragments in a very predictable way. Vectors What is the benefit of being able to produce DNA fragments? To fully understand this, we need to introduce the concept of the DNA vector. Vectors are segments of DNA that are replicated in living cells, and have been engineered to allow the insertion of other DNA fragments (like a gene, for example). An example of a vector that we've seen before is a plasmid. There are many types of vectors, including virus DNA, plasmid/virus hybrids, and artificial eukaryotic chromosomes, but plasmids are the most common and easiest to understand. The insertion of DNA into a vector is done by cutting both the vector and DNA of interest (the "insert") with the same restriction enzyme. The sticky ends on each piece of DNA will be complementary to each other, due to the palindromic nature of the restriction enzyme recognition sites. Such DNA Molecules in solution will transiently interact with each other, and join together via hydrogen bonds formed between sticky ends, forming a recombinant DNA moledcule. Four hydrogen bonds are not strong enough to hold these molecules together for more than a few milliseconds, however, and unless the phosphodiester backbones of the DNA molecules are joined together, the two molecules will quickly pull apart. Phosphodiester backbones can by joined together by DNA ligase, which we learned of in the unit on DNA replication. If DNA ligase is present in the mixture with the two different fragments of DNA, any transiently-forming recombinant DNA molecules can be joined permanently to each other. This process is outlined in the diagram below:

Plasmids have several features that allow them to work well as vectors. Some of these features occur naturally in many plasmids whereas others have been engineered into the plasmids to make them more convenient to use. (You don't need to worry about which features are natural and which are engineered.) These features include:

1. An origin of replication, so that the plasmid DNA can be replicated in bacteria. (For more on origins of replication, see the 2.
module on DNA replication.) A selectable marker. This is a gene that allows bacteria containing the plasmid to be distinguished from bacteria that lack the plasmid. Often, the marker is a gene that provides the bacteria with resistance to a particular antibiotic. That way, if a culture of bacteria is treated with the antibiotic, those with the plasmid will live, and those without it will die. The result is a pure culture of plasmid-containing bacteria. Unique restriction enzyme recognition sites, so that the plasmid can be cut open and accept DNA inserts. There need to be uniques sites, so that the plasmid is cleaved only once, instead of being cut into multiple pieces, which would make the vector useless. Often, unique restriction enzyme sites are grouped together in one region of the plasmid called a multiple cloning site or polylinker.

3.

Once created, these recombinant DNA molecules are inserted into bacteria by a process known as transformation, and those bacteria successfully transformed with the DNA can be selected using the selectable marker. These transformed bacteria can then be grown up

in great quantities, so that large amounts of the DNA can be recovered. This process, known as DNA cloning, is used for storage and amplification of recombinant DNA molecules. (For more on transformation, see the module on Recombination in Bacteria.) DNA Libraries In the preceding example, a known DNA molecule was inserted into a plasmid vector. Often, however, molecular biologists do not yet posess the particular DNA they are interested in - they must isolate it from a large pool of DNA molecules. To do this, a modified form of the DNA cloning scheme is utilized - the DNA library. DNA libraries are mixtures of different restriction enzyme-digested DNA molecules ligated into vectors. Libraries are constructed using the same general approach outlined above, except that instead of inserting one type of DNA fragment into the vector, there are thousands or even millions of different DNA fragments inserted into vector molecules. There are essentially two types of DNA libraries, based on the source of the DNA:

1. Genomic DNA Libraries - These libraries are made from genomic DNA (all of the DNA found in the organism's nuclei).
Genomic DNA molecules are very large (each chromosome in the nucleus is one such DNA molecule), so they must be fragmented into small enough pieces to insert into vectors. This is typically done through digestion with one or more appropriate restriction endonucleases, mechanical shearing, or a combination of the two processes. The DNA is then ligated into the vector, which could be a plasmid, but is more often a cosmid or a viral chromosome. cDNA Libraries - These libraries are made from cDNA, which are DNA copies of mRNA molecules. To make cDNA, mRNA is isolated from a tissue or whole organism, and DNA is copied from the mRNA template using an enzyme called reverse transcriptase. This enzyme works like a DNA polymerase, except that is uses RNA as a template instead of DNA. The resulting cDNA molecules are then engineered so that they have restriction enzyme recognition sites at each end of every molecule, which allows them to be digested and inserted into a vector as outlined previously.

2.

The difference between these two libraries is the nature of the DNA found in the library. A cDNA library, because it is derived from mRNA molecules, will only contain DNA representing transcribed genes. A genomic library will also contain gene sequences, but will also contain a lot of DNA that is not genes, because genes typically make up only about 1 - 5% of the total genomic DNA. However, this type of library will contain regulatory DNA sequences (such as enhancers and promoter sequences) that would not be present in a cDNA library. Therefore, the choice of library type depends on what type of DNA sequence a researcher wishes to study.

So how does someone find a specific fragment of DNA in a library? There are several ways this can be done. For example, screening cDNA libraries can be done using antibodies against the protein product encoded by the gene of interest (if such an antibody is available). Some vectors used to create libraries allow the inserted DNA to be transcribed after transformation into bacteria - these vectors have promoters built in, and since cDNA is copied from mRNA, it is capable of encoding protein. The transcripts would then be translated into protein in the bacteria. Bacteria transformed with the library can be grown in duplicate copies on plates and on filters in a way that individual bacterial colonies are produced, with each colony originating from a single bacterial cell, so all bacteria in a colony would contain the same recombinant DNA molecule, and therefore produce the same protein. Many thousands of colonies can be grown on a series of filters, and the bacteria lysed (broken open), liberating the protein, which would then adhere to the filter. Filters can be exposed to antibodies that are labeled in some way (usually with a fluorescent or radioactive tag); the antibody to the protein of interest will bind only to the colony that contained the recombinant DNA molecule encoding that protein. Once the colony is identified, the duplicate colony from a bacterial plate can be picked, and grown up to produce more of that DNA. This method doesn't work well with genomic libraries, because most recombinant clones in a genomic library do not encode protein. Another way to screen a library is with a piece of DNA similar to the DNA of interest, such as a similar gene from a different organism, for example. This strategy takes advantage of the ability of DNA to denature and renature (for a description of these properties, see the unit on Nucleic Acid Structure). In this case, transformed bacterial colonies are grown in duplicate on plates and filters, as in the previous example. The bacteria on the filters are lysed, and the DNA is denatured by treating with an alkaline solution. The filters are then exposed to a solution containing a single-stranded DNA 'probe' (the similar DNA sequence mentioned above), labeled with a radioactive or chemical tag, under conditions that promote renaturation. If the probe DNA is in excess, sequences strongly related to the probe will renature with the probe, and can be detected by exposure to X-ray film. As with the antibody approach, the corresponding colony on the plates can be picked and isolated. All of the bacteria in this colony descended from a single bacterial cell, and therefore they all contain the same genetic complement. This means that the plasmid (or whichever vector was used) in every bacterial cell in the colony will contain the same insert. Once isolated, a bacterial colony can be inoculated into bacterial growth medium, which provides the bacteria with the necessary nutrients for fast reproduction. In a matter of hours, the total number of bacterial cells can be increased over one billion-fold. The plasmid (or other vector) DNA can then be isolated from the bacteria, producing a sizable yield. In this way, it is possible to amplify the amount of a specific sequence of DNA you have to work with many times over. Bacteria from the growth culture can also be stored frozen at -70C, and will remain viable for many years. Therefore, once you have selected a particular clone from a library, you can store it and readily produce more of the DNA of interest for years without having to rescreen the library for that sequence. Polymerase Chain Reaction In the late 1980's, an alternative technique was developed by which DNA can be amplified many times over. This technique does not require the use of living bacteria or other cells, but it does require that you know the base sequence of the DNA that you want to amplify (we'll see how to determine the base sequence shortly) . This alternate technique is essentially a DNA replication reaction done in a test tube. Recall the needs of a DNA replication reaction: 1. 2. 3. 4. 5. There needs to be a DNA template. There needs to be a DNA polymerase. There need to be free deoxynucleotide triphosphates (dNTPs). The DNA template needs to be separated (denatured, unwound). There needs to be a primer for DNA polymerase to add free nucleotides to.

It is fairly straightforward to combine all of the ingredients in a small test tube and get replication to occur. For primers, as long as you know the sequence of the DNA being replicated, you can chemically synthesize a short piece of single stranded DNA (known as an oligonucleotide, or "oligo" for short) that has a base sequence complementary to the template strand and will work as a primer.

If you make two oligo primers, one complementary to each template strand, you can replicate both strands of DNA, doubling the amount of DNA.

To denature the template DNA, all you have to do is heat it up. Then to get the primers to anneal (bind), cool the reaction down. As long as the primers are in excess, they will bind preferentially to the opposite DNA template strand. Then DNA polymerase can extend the primers and synthesize new DNA. If you do this once, as shown above, you have twice as much DNA as you started with. Do it a second time, and you wind up with four times as much DNA as you started with. A third time, and you have eight times as much DNA, and so on. In other words, the DNA amount is increased exponentially. Thirty cycles will give you approxiamtely one billion times as much DNA as you started with, and when automated, it only takes about three hours! There's just one problem with this scheme. The high temperatures required to denature the DNA (near boiling temperature) also denature the DNA polymerase, rendering it inactive. You could add fresh DNA polymerase to the reaction after each denaturing step, but that would be somewhat tedious, and would interfere with automation of the procedure. So what to do? The rather ingenious solution (which won a Nobel Prize) was to use DNA polymerase isolated from bacteria that live near undersea volcanic vents at temperatures that exceed the boiling temperature of water (the water doesn't boil because it's under extremely high pressure). These bacteria have proteins that are extremely stable at high temperatures (they have to be, or the bacteria wouldn't be able to survive under those conditions), so their DNA polymerases can stand the denaturation temperature of the reaction described above. The bacterial species used is called Thermus aquaticus, and using the same convention as that for naming restriction enzymes, the enzyme is called Taq DNA polymerase. The use of Taq DNA polymerase allowed automation of this process, known as polymerase chain reaction, or PCR, and it revolutionized molecular genetics by allowing rapid amplification of any DNA of interest. The technique has also revolutionized forensic science, because it allows amplification of DNA from minute samples such as blood droplets, which then allows DNA fingerprints to be performed. Analysis of Cloned DNA Sequences Once DNA has been cloned and amplified, it needs to be analyzed and characterized. Two common ways to do this are restriction enzyme mapping (and its variation, the Southern Blot) and DNA sequencing, both of which rely upon a technique known as gel electrophoresis. Gel electrophoresis takes advantage of the overall negative charge possessed by nucleic acids, due to the phosphates in the backbone. Because nucleic acids are negatively charged, they will migrate in an electric field from the negative electrode to the positive electrode (because like charges repel each other). If the electric field and the nucleic acids are run in a semisolid matrix (i.e. a 'gel' usually composed of agarose, a gelatin-like matrix, or polyacrylamide, a chemically cross-linked meshwork), then the nucleid acids will migrate at a speed inversely proportional to their size. In other words, large molecules will migrate slowly, because they will be held

up by the gel, and small molecules will migrate quickly, because they can pass more easily through openings in the meshwork of the gel. Practically speaking, a mixture of DNA molecules can be loaded into a depression in a gel (the depression is called a 'well'), the gel is surrounded by an electrolyte solution, and electric current is passed through the gel. The fragments of DNA in the mixture will separate out according to size, and can be visualized using a DNA-specific stain, such as ethidium bromide, which binds to DNA and glows orange under UV light.

Restriction enzyme mapping entails digesting the DNA interest with various restriction enzymes, singly and in pairs. By running the various reactions on agarose gels, it is possible to determine the sizes of the digested fragments (by comparing to DNA standard fragments of known size). This in turn allows mapping of the different restriction enzyme recognition sites relative to each other. For more on this process, see the Restriction Mapping animation on the Russell CD-ROM. Sometimes, it is necessary to visualize only a few restriction fragments out of a large set of fragments. To do this, a technique known as Southern blotting (named after the man who developed the technique) is used. Southern blotting involves taking an agarose gel after the separation of DNA fragments, denaturing the DNA (using high pH), placing the gel against a special type of filter paper (usually nylon or nitrocellulose), and using a flow of liquid to move the DNA fragments vertically out of the gel and onto the filter. The filter can then be incubated with a labeled probe, just as was done during library screening. The probe will anneal to complementary sequences, and those sequences that anneal can be visualized because of the label on the probe.

(As an aside, the same technique can also be applied to RNA. The RNA is separated on an agarose gel, transferred to a filter, and annealed to a probe. In this way, a single species of RNA can be visualized out of a mixture of thousands of different RNA molecules. This techniques, because it is an adaptation of Southern blotting, is known as Northern blotting.) DNA Sequencing Ultimately, to properly study and utilize a piece of DNA, we need to know its sequence of bases. This can be done using a technique called DNA sequencing, which utilizes a modified DNA replication reaction. As with other DNA replication reactions, this one requires a primer. For DNA sequencing, the primer is often complementary to a sequence within the vector, close to where the DNA of interest is inserted. To visualize the synthesized DNA, the primer is labeled, either radioactively or with a fluorescent tag. Other components of the reaction are normal components of a DNA replication reaction, except for one. One of the nucleotides is a dideoxynucleotide triphosphate (ddNTP). This means that in addition to lacking a hydroxyl group at the 2' position of the sugar, it also lacks a hydroxyl group at the 3' position of the sugar. Think about what this would mean to DNA replication. Where does DNA polymerase add a free nucleotide to on the growing polynucleotide chain? That's right - to the 3' hydroxyl of the nucleotide at the end of the chain. If the nucleotide at the end of the chain has no 3' hydroxyl (if it is a ddNTP), then there is no place for DNA polymerase to add another nucleotide and DNA synthesis will stop. So how does this help determine the sequence of the bases? Imagine the following reaction: a standard DNA replication reaction with a template (the DNA being sequenced), a primer (labeled), DNA polymerase, all four dNTPs, and one ddNTP (for example, let's say it is ddATP) at a low concentration. Because the ddNTP is at a low concentration, it will be inserted into the DNA chain only occasionally and randomly - sometimes in the first A position, sometimes in the second A position (with a normal A in the first position), sometimes in the third A position, etc. The result would be a mixture of labeled DNA molecules, all terminating at different positions, but all ending with A. If this reaction is run on a gel, it will produce a ladder of bands. Now extend this by adding three more separate reactions, each with a different ddNTP. There will be one reaction with fragments all ending in C, one with fragments all ending in G, and one with fragments all ending in T. Each of these is run on an adjacent lane in the gel, and the sequence can be read right off the gel, because longer fragments will not run as far as smaller fragments, so the fragments can be ordered in terms of size, and we know the base that ends each fragment.

For a nice presentation of the process of DNA sequencing, check out the DNA sequencing animation on the Russell CD-ROM. Expression of Cloned Genes Ultimately, the main reason we clone and characterize genes is so we can produce the protein. This is done for a variety of reasons. Sometimes, the protein is a product that will be harvested for commercial sale (for example, by a biotechnology or pharmaceutical company). Production of the protein can also be used in an indirect way to understand its function. By expressing the gene in cells or an organism (for example, expressing a gene at a time or in a place that it wouldn't normally be expressed), it may be possible to observe the effect that expression has on the organism, and infer the function from that. So how do you express a gene? There is a class of vector known as an expression vector. These vectors have promoters (often from viral genes) engineered into the vector near the multiple cloning site. Any gene inserted into the multiple cloning site therefore comes under the control of the promoter, allowing expression of the gene under appropriate conditions. Expression may be in vivo (in a living cell or organism) or in vitro (in a test tube using cell-free transcription and translation systems that are available commercially, allowing production of the protein relatively purely). Techniques of Molecular Genetics: Summary of Key Points Restriction endonucleases are enzymes that recognize and cleave specific palindromic sequences in DNA. DNA segments of interest may be isolated by "cloning" - cutting the DNA with a restriction enzyme, and ligating it into a vector, such as a plasmid, which can then be reproduced in bacteria. DNA libraries are bacterial or viral cultures that contain mixtures of DNA molecules inserted into vectors. Polymerase Chain Reaction (PCR) is an alternate technique for the amplification of DNA. This technque requires knowledge of the sequence of the DNA to be amplified. Once cloned, DNA fragments can be characterized by restriction enzyme mapping, by DNA sequencing, or by gene expression studies.

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells. 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 may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid. 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. Several different transfection techniques are available, such as calcium phosphate transfection, electroporation, microinjection and liposome transfection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. 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. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. 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. Polymerase chain reaction (PCR) The polymerase chain reaction 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 can also be used to determine whether a particular DNA fragment is found in a cDNA library. 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. Gel electrophoresis Gel electrophoresis is one of the principal tools of molecular biology. 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. Macromolecule blotting and probing The terms ''northern'', ''western'' and ''eastern'' blotting are derived from what initially was a molecular biology joke that played on the term ''Southern blotting'', after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the ''northern blot'' actually didn't use the term. 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.

Southern blotting Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a 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. Most original protocols used radioactive labels, however nonradioactive alternatives are now available. 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. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines. 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. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. 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 intensity of these bands is related to the amount of the target RNA in the samples analyzed. 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. Western blotting Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques. 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. Often, the antibodies are labeled with an enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, these ''immunostaining'' methods, such as FISH, are used more often in cell biology research. 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. Arrays A DNA 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. Arrays make it possible to put down a large quantity of very small (100 micrometre diameter) spots on a single slide. 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 the exact 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. For instance, the common baker's yeast, ''Saccharomyces cerevisiae'', contains about 7000 genes; with a microarray, one can measure qualitatively how each gene is expressed, and how that expression changes, for example, with a change in temperature. There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array. 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. Allele Specific Oligonucleotide Allele specific oligonucleotide (ASO) 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. 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.