Discovery of Controlled Swarm in Bacteria

A study led by researchers from Universitat Autònoma de Barcelona (UAB) describes one of the mechanisms in which pathogenic bacteria populations control the way they spread over the surface of the organs they infect and stop when they detect the presence of an antibiotic, only to resume again when the effect wears off. The star of this process is the RecA protein, which significantly increases its concentration at the start of the bacteria DNA repair mechanism induced by antibiotics.

The research was published in Infection and Immunity.

In order to develop the infectious process, many pathogenic bacteria move collectively along the surface of the organ they infect until growing into a massive colony, and consequently produce toxins and substances that harm host tissues. This movement is known as swarming, similar to the movement of bee colonies and other animals. Parts of the molecular process taking place during this movement already have been described, but the mechanism controlling activation or inhibition was not yet known.

The research reveals for the first time the relation between the bacteria DNA repair system, known as SOS response, and swarming. Researchers demonstrated that the presence of antibiotics activates SOS response and thus increases concentration of RecA protein. This interferes with the action of the CheW protein, essential for swarming, and thus causes the bacterial colony to stop moving. When the concentration of this antibiotic decreases, the amount of RecA protein reduces and CheW once again can continue its task of spreading the bacteria.

The results obtained indicate that given the special characteristics of this type of collective movement, antibiotics only affect outer cells of the swarm, which in turn act as sensors and activate the aforementioned molecular repair system. This action thus cancels out the effect of the drug on the rest of the bacteria population.

Jordi Barbé, Laura Medina Ruiz and Susana Campoy, researchers at UAB's Department of Genetics and Microbiology and directors of the study, highlight the importance of this basic discovery since it will allow for the design of targets blocking RecA action and thus increase antibiotic sensitivity of bacteria.

The research was carried out with Salmonella enterica, member of a bacteria group found in several pathogenic species responsible for diseases in the digestive and respiratory system, such as septicaemia and systemic infections.

Working alongside UAB researchers were Cristina Latasa of the Institute for Agrobiotechnology-Public University of Navarre-CSIC-Government of Navarre, and Paula Cárdenas and Juan Carlos Alonso of the National Biotechnology Centre belonging to the Spanish Higher Council for Scientific Research (CSIC).

Gene Cloning And DNA Analysis- An Introduction By T.A. Brown

Known world-wide as the standard introductory text to this important and exciting area, the fifth edition of Gene Cloning and DNA Analysis addresses new and growing areas of research whilst retaining the philosophy of the previous editions. Assuming the reader has little prior knowledge of the subject its importance, the principles of the techniques used and their applications are all carefully laid out, with over 250 clearly presented two-colour illustrations.
In addition to a number of informative changes to the text throughout the book, the final four chapters have been significantly updated and extended to reflect the striking advances made in recent years in the applications of gene cloning and DNA analysis in biotechnology:

• Extended chapter on agriculture including new material on glyphosate resistant plants
• New section on the uses of gene cloning and PCR in archaeology
• Coverage of ethical concerns relating to pharming, gene therapy and GM crops

Gene Cloning and DNA Analysis remains an essential introductory text to a wide range of biological sciences students; including genetics and genomics, molecular biology, biochemistry, immunology and applied biology. It is also a perfect introductory text for any professional needing to learn the basics of the subject. All libraries in universities where medical, life and biological sciences are studied and taught should have copies available on their shelves.
Terry Brown is Professor in the Faculty of Life Sciences, The University of Manchester, Manchester, UK 

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Developing nanotechnology-based cancer therapeutics

Using nanoscale particles loaded with chemotherapeutic agents – and covered with homing molecules that allow them to selectively bind to cancer cells – our investigators are building a new class of "smart bombs" for cancer. These functionalized nanoparticles hold the promise of both reducing toxicity and improving efficacy of a wide range of existing anti-cancer agents. Nanotechnology is also being applied to RNA interference (RNAi), which can be used to silence genes in many biological systems. RNAi could be used to inhibit the function of virtually any cancer-causing gene.

be used to inhibit the function of virtually any cancer-causing gene.

Research Summary The goal of this area is to develop cancer therapeutics for the 21st century based on nanotechnology. Current therapeutic agents for cancer are small molecule drugs that either damage DNA or target intracellular proteins and antibodies that bind to and block the activity of extracellular proteins. Some of these agents work effectively for certain cancer types; however, all too often, cancer therapies are woefully inadequate.

The emerging generation of molecularly targeted anti-cancer agents, contributed to significantly by MIT investigators, has improved treatment in select cancer types to date. Despite this progress, there is a pressing need to develop even more powerful approaches to cancer therapy, indeed to establish entirely new paradigms for cancer treatment. For example, using nanoscale particles loaded with conventional chemotherapeutic agents and covered with homing molecules that allow them to selectively bind to and enter cancer cells, our investigators have built a new class of "smart bombs" for cancer. Preliminary tests in pre-clinical models have given very encouraging results to date.

These functionalized nanoparticles hold the promise of both reducing toxicity and improving efficacy of a wide range of existing anti-cancer agents. Clinical trials in humans are already being contemplated with this first class of nanoscale therapies. Future work in this program will focus on the generation of nanoparticles that can literally seek out cancer cells wherever they might be in the body and specifically deliver their payloads at high doses, thus eliminating the cancer cells without causing unwanted tissue damage.

Nanotechnology is also being applied to develop the promising new field of RNA interference (RNAi) for therapeutic purposes. Discovered only in the last decade, RNAi is a powerful tool that can be used to silence any gene of interest in many biological systems. To date, RNAi has been used largely for research applications, involving cells grown in the laboratory or model organisms. Multi-disciplinary teams of biologists and engineers at MIT are working together to move this technology forward so that it can be used one day to inhibit the function of cancer genes for cancer therapy.

Our goal is to develop improved delivery methods such that RNAi molecules can be given safely and effectively in cancer patients. Again, targeting the RNAi molecules selectively to cancer cells will likely be required to achieve the desired therapeutic index. Similar approaches are being developed for delivering therapeutic DNA molecules to cancer cells in vivo, including to replace the function of mutated tumor suppressor genes.

How Scientists Separate Proteins

Abstract

Have you ever had to sort a jumble of objects into piles, based on their type? Maybe laundry, or a big load of dishes, or while organizing the garage or a closet? Scientists have to do something similar when they want to study or isolate just a single type of protein. For example, a botanist might discover an exotic plant that is poisonous when eaten, but that also has great antibiotic properties. To help develop a new antibiotic for human use, he or she would have to separate the different plant proteins, and research which ones are toxic and which ones help fight off infections. One way scientists do this is by sorting the proteins, based on their size, using a technique called size-exclusion chromatography. In this science fair project, you can try your hand at this biochemical sorting technique and use it to determine the relative size of the green fluorescent protein (GFP).

Objective

To determine the relative size of green fluorescent protein (GFP), using size-exclusion chromatography.

Introduction

Manufacturing a life-saving drug, studying how the APOE protein contributes to Alzheimer's in humans, researching the effects of a bacterial toxin—these are all real-life instances where a biologist or a biochemist would need to separate a mixture of proteins. How are proteins separated? There are several methods, but one of the simplest ways is size-exclusion chromatography, which uses the size of a protein as a physical means of sorting. Different proteins come in different sizes, much like a grapefruit, an orange, and a lime are all citrus, but you can tell them apart by size alone.

In size-exclusion chromatography, a protein mixture, suspended in a special liquid, is poured into a column. The column, as shown in Figure 1 below, contains a suspension of tiny beads with different-sized macroscopic pores. As the proteins travel through the column, they get tangled in the beads. The larger proteins can fit in fewer pores and thus, have a more direct and quicker path through the beads. In contrast, the smaller proteins can fit through more pores and take a more meandering path as they get caught in various holes and take a longer time to flow through the column. As time passes, the special liquid, called an eluate, drips out of the column and into tubes. The initial drips of eluate contain the larger proteins, which passed through quickly, while the later drips contain the smaller proteins. Changing the collection tube under the column every couple of minutes results in multiple tubes of protein—each tube containing proteins of a different sizes.

Figure 1. Size-exclusion chromatography columns contain beads with various-sized pores. The larger proteins, which fit in fewer pores, take a more direct path through the column and elute first. The smaller proteins are slower to elute as they meander in and out of pores.

In this science fair project, you'll use size-exclusion chromatography to approximate the size of green fluorescent protein (GFP). GFP, first isolated from the jellyfish Aequorea Victoria, glows green when exposed to blue light. Because of its fluorescent properties, it is often used in cellular and molecular biology to track the expression of genes and the location of other proteins. In such tracking studies, it is important to keep the size of GFP in mind—if it is too large, it might not be able to move into some cellular areas, thus skewing the results of the study. To determine the size of GFP, you'll used a protein size-exclusion chromatography kit from Bio-Rad, which is explained in the Materials and Equipment section, below. The kit contains two other proteins: hemoglobin and vitamin B₁₂. You'll create a mixture of hemoglobin, vitamin B₁₂, and GFP, then sort them with the chromatography column. Since hemoglobin is reddish-brown in color and vitamin B₁₂ appears pink, you'll be able to tell which eluate samples contain each of these proteins. Using a UV light to make the GFP fluoresce, you'll determine which eluate samples contain GFP. From this information, you'll be able to determine the size of GFP, relative to the sizes of hemoglobin and vitamin B₁₂.

Terms, Concepts and Questions to Start Background Research

• Size-exclusion chromatography
• Eluate
• Green fluorescent protein (GFP)
• Hemoglobin
• Vitamin B₁₂
• Kilodalton

Questions

• How does size-exclusion chromatography work?
• What are the sizes, in kilodaltons, of hemoglobin and vitamin B₁₂?
• From where does GFP come?
• What other methods can be used to separate protein mixtures? What are their advantages and disadvantages?

Bibliography

• Tissue, B.M. (2000). Size-exclusion Chromatography (SEC). Retrieved July 28, 2009, from http://www.files.chem.vt.edu/chem-ed/sep/lc/size-exc.html
• Zimmer, M. (2009, June 21). GFP: Green Fluorescent Protein. Retrieved July 28, 2009, from http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
• Wikipedia Contributors. (2009, July 24), Protein Purification. Wikipedia: The Free Encyclopedia. Retrieved July 28, 2009, from http://en.wikipedia.org/w/index.php?title=Protein_purification&oldid=303904910
• Wikipedia Contributors. (2009, July 28). Size-exclusion Chromatography. Wikipedia: The Free Encyclopedia. Retrieved July 28, 2009, from http://en.wikipedia.org/w/index.php?title=Size_exclusion_chromatography&oldid=304671149

Materials and Equipment

• Pipette, capable of dispensing 4-ml volumes
• Micropipettes, capable of dispensing 5- to 250-ul volumes
• Micropipette tips
• UV light
• Bio-Rad Size-exclusion Chromatography Kit from http://www.bio-rad.com
• Green fluorescent protein (GFP) sample
  1. You may be able to obtain this from a science teacher, or from a researcher at a local university. If not, you can use the Bio-Rad pGLO Bacterial Transformation from http://www.bio-rad.com and Green Fluorescent Protein Chromatography Kits from http://www.bio-rad.com to go through all the steps of creating a laboratory sample of GFP.
• Lab notebook

Experimental Procedure

Note: The Bio-Rad kit includes excellent detailed instructions, thus the procedure below is just an outline of the experiment. For step-by-step technical instructions, please consult the kit manuals.

Creating the Protein Mixture Sample

1. If you need to create your own GFP sample:
   1. Follow the instructions that came with the pGLO Bacterial Transformation kit to put the GFP gene into bacteria (i.e. transform the bacteria).
   2. Then use the Green Fluorescent Protein Chromatography Kit and the instructions contained therein to grow the GFP expressing bacteria, and purify the protein.
2. Once you have your GFP sample (either donated to you or created with the kits in step 1), combine 5 ul of the GFP protein sample with 10 ul of the hemoglobin/vitamin B₁₂ mixture from the Size-exclusion Chromatography Kit. This new 15-ul mixture will be your protein mixture sample for the size-exclusion chromatography experiment.

Performing the Size-exclusion Chromatography and Analyzing the Results

1. Carry out the size-exclusion chromatography, as instructed in the Bio-Rad kit's manual, using the GFP/ hemoglobin/vitamin B₁₂ mixture as your protein sample.
2. In the end, you should have 10 collection tubes from the columns. Note the color, in natural light, of each of these 10 tubes. Write your observations in your lab notebook.
3. Based on your visual inspection and background reading, which tubes contain hemoglobin? Which collection tubes contain vitamin B₁₂? Which tubes do you expect to contain GFP?
4. Look at all 10 collection tubes in a dark room, using only a UV light. Tubes containing GFP will glow green under the UV light. Record which tubes contained GFP and the relative brightness (which corresponds to the relative quantity of GFP) in each tube. Do your observations match your expectations?
5. Based on the results of the size-exclusion chromatography, what can you conclude about the size of GFP? Is it larger or smaller than hemoglobin? How about vitamin B₁₂? Can you make any guesses about its size, in kilodaltons?

Variations

• Size-exclusion chromatography can only help you determine a relative size for GFP. Devise a way to get a better estimate, in daltons, of the size of GFP.
• Try adding other proteins to the mixture and figuring out ways to isolate each one.
• How does pH affect size-exclusion chromatography? Devise an experiment to find out.

A Magnetic Primer Designer

Abstract

How do scientists "copy" DNA? They use a process called the Polymerase Chain Reaction, or PCR. The key to making this process work is having a primer that will stick to the piece of DNA you want to copy, called a template. In this experiment you will test how the number of matches and mismatches in a primer will affect its ability to stick, or anneal, to the DNA template during PCR.

Objective

In this experiment you will test how matches and mismatches affect the ability of primers to stick to the DNA that is copied during PCR.

Introduction

All living things come with a set of instructions stored in their DNA, short for deoxyribonucleic acid. Whether you are a human, rat, tomato, or bacteria, each cell will have DNA inside of it. DNA is the blueprint for everything that happens inside the cell of an organism, and each cell has an entire copy of the same set of instructions. The entire set of instructions is called the genome and the information is stored in a code of nucleotides (A, T, C, and G) called bases. Here is an example of a DNA sequence that is 12 base pairs long:

Notice that this piece of DNA has two sequences: one on the top, and one on the bottom. DNA is double stranded, which means that it has two strands. The nucleotides of each of these strands are paired together in a particular way to match the other strand: A pairs with T and C pairs with G. If a nucleotide is paired according to these rules, it is called a match. But if the nucleotide is not paired properly, then it is called a mismatch. Matches and mismatches can affect how the two strands of DNA pair together, and sometimes a mismatch can lead to a mutation.

The information stored in the DNA is coded into sets of nucleotide sequences called genes. Each gene has a set of instructions for making a specific protein. The protein has a certain job to do, called a function. Since different cells in your body have different jobs to do, many of the genes will be turned on in some cells, but not others. For example, some genes code for proteins specific to your blood cells, like hemoglobin. Other genes code for proteins specific to your pancreas, like insulin. Even though different genes are turned on in different cells, your cells and organs all work together in a coordinated way so that your body can function properly.

What if there is something wrong with one of your genes? This can cause problems for your body and how it functions. For example, people who have type I diabetes have problems making insulin. To help people with diabetes, scientists figured out a way to make insulin that diabetics can inject into their body. The insulin is made by a bacteria that has the human gene for insulin.

For scientists to study a gene, they need to be able to isolate it. The simplest way to isolate a gene is to clone the gene into a bacteria, but first you need many, many copies of the gene you want to clone. How do you make copies of DNA? The Polymerase Chain Reaction (PCR) makes copies of a DNA template in four main steps:

• Melt - a high temperature (95^oC) will melt (separate) the two strands of the DNA you want to copy (called the template)
• Anneal - the primer will stick to (anneal to) the template strand
• Extend - the enzyme will copy (replicate) the template DNA strand starting with the primer
• Repeat - the process will be repeated over and over, usually about 30 cycles

PCR is like a copy machine for DNA! (Copyright © The Royal Swedish Academy of Sciences, 2003)

If these first three steps are repeated 30 times, a scientist can make 1 billion copies of a single piece of DNA! That provides the scientist with plenty of DNA material to clone and study. PCR is a very important step in the discovery and manufacturing of genes that become important pharmaceuticals, like the insulin gene.

Notice that if the primer doesn't stick, then the enzyme won't have a place to start copying the template DNA, so designing a good primer is a very important first step for PCR success. In this experiment you will build a model of a primer sticking (annealing) to a DNA template strand using magnets. How important is it for the strength of the primer for the sequence of the primer to match the template strand? You will make matches (where the magnets will stick to each other) and mismatches (where the magnets will repel each other) to model nucleotides in the primer annealing to the DNA template. Will more matches make the primer stick better than a primer with mismatches?

Terms, Concepts and Questions to Start Background Research

To do this type of experiment you should know what the following terms mean. Have an adult help you search the Internet, or take you to your local library to find out more!

• DNA sequence
• Polymerase Chain Reaction (PCR)
• Nucleotide (A, T, C, G)
• Match
• Mismatch
• Anneal

Questions

• How do scientists make copies of DNA?
• What does a primer do, and how does it anneal?
• How will matches or mismatches affect the ability of the primer to anneal to the DNA of interest?

Bibliography

• Check out this site from the Cold Spring Harbor Laboratory for two animations of DNA being amplified and to watch interviews with the inventor of PCR, Kary Mullis:
  DNAi, 2003. "DNA Interactive: Manipulation: Techniques: Amplifying" DNA Interactive (DNAi), Dolan DNA Learning Center, Cold Spring Harbor Laboratory, NY. [accessed March 6, 2007] http://www.dnai.org/b/index.html
• Half of the 1993 Nobel Prize for Chemistry was awarded to Kary Mullis for his invention of the polymerase chain reaction (PCR) method. Try playing this game at NobelPrize.org to learn more about how PCR works:
  Backman, A., 2007. "The PCR Method: A DNA Copying Machine," NobelPrize.org [accessed March 6, 2007] http://nobelprize.org/educational_games/chemistry/pcr/index.html
• Learn what DNA is and what is does from the Genetic Science Learning Center:
  The Tech, 2004. "What is DNA?" The Genetic Science Learning Center, University of Utah, Salt Lake City, UT. [accessed March 6, 2007] http://learn.genetics.utah.edu/units/basics/tour/dna.swf
• Visit this online exhibit at The Tech Museum of Innovation to understand and visualize where the DNA in your body is: Rosa, C. et. al. 2007. "Zooming Into DNA". The Tech Museum of Innovation, San Jose, CA. [accessed March 6, 2007] http://www.thetech.org/genetics/zoomIn/index.html

Materials and Equipment

• Small, flat magnets like the Master Magnetics Ceramic Disc Magnet Value Pack (Model# 07049, 51 Pieces)
  • Available for order online from Amazon:
    http://www.amazon.com/Master-Magnetics-Ceramic-Magnet-Value/dp/B000IYKKV2
  • You can also buy them at a local hardware store like ACE or Home Depot.
• Clear packaging tape, 2 inches wide
• Paint pen in a light color (yellow or white for example)
• Hole punch
• Paper clip
• Dixie cup
• Pennies
• Metric ruler

Experimental Procedure

1. Arrange the magnets in a stack to identify the north and south pole ends of each magnet. Remember that opposites attract, so your magnets will be arranged in an opposing order: N/S, N/S, N/S, etc.
2. Indicate which sides have similar poles by painting the corresponding side of each magnet with a paint pen. When you arrange the magnets in a stack again, the colors should be alternating. (If the magnets you are using already have the poles indicated, then you can skip this step.)
3. The painted side of each magnet will be designated + (plus) and the unpainted side will be - (minus).
4. Next, you will make a simplified model of a DNA strand that you will copy using PCR.
5. Lay out a strip of clear packaging tape (20 cm long), sticky side up, on a table.
6. Place ten magnets along one side of the tape with the painted sides up, spacing the magnets 2 cm apart. The sequence for the DNA strand will be either all plus (+ + + + + + + + + +) OR all minus ( - - - - - - - - - - ) depending upon which side is facing forward. In the example below, let's assume the strand is facing with the negative side towards you (the all minus side).
7. Fold the tape over the magnets, creating a long strip of magnets embedded inside the clear tape. Your model DNA strand should look like this:

8. Now you are ready to make your "primers" which will be shorter versions of your DNA model. Each primer will be 5 magnets in length, but the sequence of the primers will be different.
9. Place a strip of clear packaging tape (10 cm long), sticky side up, on a table.
10. Arrange 5 magnets along one side of the tape, alternating the poles of the magnets in any order you choose. It is important for the positions of the magnets in your primer to be in the same places as the magnets in your DNA strand. Your model primer strand should look like this:

11. Repeat, making more primers with different sequences. It is important to include a positive control primer (+ + + + +) and a negative control primer ( - - - - - ) which can actually be the same primer flipped over!
12. Write the sequences of the primers in a data table, here is an example:

    Primer Name	Primer Sequence	Number of Matches	Number of Mismatches	Number of Pennies
    Positive	+ + + + +	5	0
    Primer 1	+ + + + -	4	1
    Primer 2	+ + + - -
    ...
    ...
    Negative	- - - - -	0	5

13. To test the strength of each primer, you need to add weight to the primer while it is sticking (annealing) to the DNA strand. Then you will increase the weight until the primer falls off. You will do this by attaching a small paper cup to the end of the primer and adding pennies.
14. At one end of the primer strand, use the hole punch to punch a hole near the end of the tape strip.
15. Unfold a paperclip to make a hook, and slip it through the hole of the primer strand and then through the paper cup, so that the cup will dangle from the end of the primer strand. The primer, with hook attached, should look like this:

16. Now you will stick each "primer" to the "DNA" sequence and see if the two strands anneal, or stick together. Count the number of matches and mismatches between your primer and the DNA strand. Write this number in your data table. Here is an example of a primer with one mismatch at the end. See how the magnets repel each other at the end of the strand?

    
    Here is another example of a primer with two mismatches in the middle. See how the magnets repel each other in the center causing a bulge?

17. Hold the DNA strand by one end so that the end of the primer with the paper cup attached will dangle from the other end. Add pennies, one at a time, until the primer falls off. While you are adding pennies to the cup, your experiment will look like this:

18. Write the number of pennies you added in the data table and then test the next primer. Continue testing the primers until you have tested them all. Here are the results for the first two primers tested from the examples above:

    Primer Name	Primer Sequence	Number of Matches	Number of Mismatches	Number of Pennies
    Positive	+ + + + +	5	0	50
    Primer 1	+ + + + -	4	1	33
    Primer 2	+ + + - -
    ...
    ...
    Negative	- - - - -	0	5

    TIP: You can repeat your tests for each primer several times and then calculate an average to get better, more reliable data.

19. Make a line graph of your data, placing the number of pennies on the left side (y-axis) and the number of mismatches on the bottom (x-axis) of the graph.

5 Ways To Clean Up DNA Sample

One of the most common tasks in molecular biology is cleaning up DNA from aqueous solutions to remove buffer salts, enzymes or other substances that could affect downstream applications. Examples include cleaning up PCR reactions, digests or other enzymatic treatments and cleaning up genomic or plasmid DNA contaminated with cellular proteins/debris. There are several ways to approach DNA clean-up, here are five of them.

1. Phenol/Chloroform extraction

Phenol chloroform extraction (see Kirby, 1957), normally followed by ethanol precipitation, is the traditional method to remove protein from a DNA sample. In this procedure, the DNA solution is mixed with phenol and chloroform. The water-soluble DNA partitions into the aqueous phase, while the proteins are denatured by the organic solvents and stay in the organic phase. The aqueous phase containing the protein-free DNA can then be collected.

Advantages: A cheap and effective way to remove proteins from DNA solutions
Disadvantages: Slow compared to most modern methods, there is a risk of phenol/chloroform carry-over into the final sample (which could inhibit downstream enzymatic reactions), chloroform and phenol are both hazardous chemicals.

2. Ethanol Precipitation

Ethanol precipitation is a tried and tested method for de-salting and concentrating DNA. 0.1 to 0.5 M monovalent cations (normally in the form of the acetate salt of sodium) is added to the DNA, along with ethanol to a final concentration of 70%. Ethanol changes the DNA structure so that the DNA molecules aggregate and precipitate from solution (see Eickbush and Moudrianakis, 1978). Since most salts and small organic molecules are soluble in 70% ethanol they stay in solution and the precipitated DNA can be separated from them by centrifugation.

Advantages: A cheap and effective way to de-salt and concentrate DNA.
Disadvantages: Time consuming and risk of ethanol carry-over into the final sample

3. Silica column-based kits

Column-based kits offer a convenient approach to DNA cleanup. The principle is that chaotrophic salts are added to the sample to denature the DNA by disrupting it’s hydrogen bonding. Under these conditions, the DNA will selectively bind to the silica resin in the column, allowing it to be separated from the rest of the sample. After washing the DNA is eluted from the column with a low salt solution which allows the re-naturing of the DNA, causing it to lose affinity for the silica. A good example of this technology is Qiagen’s Qiaquick series, which has several kits for agarose gel extraction, enzymatic reaction, nucleotide and PCR clean-up.

Advantages: Convenient, relatively fast and the user can process large number of samples using the vacuum manifold option.
Disadvantages: Fairly expensive, and in my experience low yields (as low as 25%) and chaotrophic salt carry-over are common.

Note: Zymo’s DNA clean-up and concentrator kit offers an alternative, based on the same silica resin technology, where the sample can be eluted in a very small volume to give high DNA concentrations.

4. Strataclean Resin

Stratagene’s StrataClean approach uses a slurry of hydroxylated silica, which (almost magically it seems) binds protein with a high affinity, while having a low affinity of DNA at near neutral pH. The slurry is added directly to the DNA sample, which is then mixed and centrifuged and the supernatant containing the protein-free DNA is collected. The protocol takes just a few minutes, although 2 or 3 clean-ups may be required to certain stubborn enzymes (details are available in the kit’s protocol).

Advantages: Very fast and cheap, no chaotrophic salts or organic washing solutions
Disadvantages: Only removes proteins. Removal of salts requires a traditional ethanol precipitation step.

5. Magnetic Beads

This approach uses magnetic beads that conditionally bind DNA and can be immobilized on a magnetic to separate the DNA from the rest of the sample and allow washing etc. Invitrogen’s ChargeSwitch technology is the best example of this I have seen. It uses magnetic beads that are positively charged, and will therefore bind to DNA, at low pH but at high pH they are negatively charged and release the DNA.

Advantages: Fast, no chaotrophic salts or organic washing solutions, very good yields and spectophotmetric purity in my experience
Disadvantages: The inital outlay for the magnets is reasonably high and the procedure is a bit tricky when handling multiple samples.