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CALTECH APH 162 - DNA Science Techniques

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Weeks 3 and 4 : DNA Science - TechniquesDave Wu and Frosso SeitaridouJanuary 9, 20071 IntroductionDuring these two weeks you will be working with DNA. Even though DNA is a very robustmolecule, there are many techniques that have been developed which facilitate the process ofworking with DNA. These techniques are, in general, very popular in biochemistry and molecularbiology. Below, we list these techniques and give a brief description for each. At the end of eachsection, we provide a source list, where more details can be found.2 PCR - Polymerase Chain ReactionPCR stands for Polymerase Chain Reaction. This is a technique used to amplify (i.e.make many copies of) specific regions of a DNA strand. The regions to be amplified are usuallysmall, around 10kb. In order for the DNA to be amplified, there are several components thatgo into the reaction:• DNA template: This, of course, is the region of the DNA to be amplified• The two primers: They determine the beginning and the end of the DNA fragment that willbe amplified. Usually, they are between 18 − 25 base pairs long. They are complementaryto the beginning or end of the DNA fragment to b e amplified. The primers is where theDNA polymerase will bind in order to start the synthesis of the new DNA strand.• DNA polymerase: This does the actual copying of the region to be amplified• dNTPs: They are the four nucleotides that the DNA consists of. In order to amplify theDNA fragment, we will need nucleotides that the DNA polymerase can use• Buffer solution: This provides the right environment for the DNA polymerase to operate(contains ions, the right pH, etc.)The PCR procedure is comp osed of several steps. We briefly describe each one of thesesteps below:1• Denaturing: In order for the DNA to be amplified, it needs to be broken apart into its twostrands (denaturing). This happens when it is heated at a high temperature, 96oC, whichbreaks apart the hydrogen bonds that connect the two DNA strands. Before the firstcycle, we denature for an extended time period (5min), to ensure that both the templateDNA and the primers are single stranded. During this step certain polumerases are alsoactivated.• Annealing: After the denaturing process, the temperature is lowered so that the primerscan anneal (i.e. attach) to the single DNA strands. The temperature of this step is nor-mally 5oC lower than the melting temperature of the primers. Normally, this temperatureis in the range 45 − 65oC. This temperature is important because if it too high, then theprimers might not bind at all and if it is too low, they will bind randomly. The durationof this step is around 30sec.• Elongation: Now that the primers are annealed, the DNA polymerase can attach to theprimer and use the dNTPs in order to synthesize the new strands, as it moves alongthe DNA strand of the template. The temperature for this step depends on the DNApolymerase and the time depends both on the polymerase and on the length of the DNAto be amplified. The latter translates to about 1 minute per thousand base pairs. Afterthe elongation is complete, the newly formed DNA strands can be used as a templatefor a second cycle of DNA amplification. Consequently, the denaturing, annealing andelongation steps can be repeated for a number of cycles (around 30 cycles) in order toobtain a large amount of DNA copies of the initial template. After the final cycle, a finalelongation time of about 10 minutes is used in order to finish copying any remaining singlestranded DNA.References:• Wikipedia: Polymerase chain reaction• http://users.ugent.be/ avierstr/principles/pcr.html• http://people.ku.edu/ jbrown/pcr.html• http://www.dnalc.org/ddnalc/resources/pcr.html3 Restriction and LigationRestriction refers to the process of using enzymes (called restriction enzymes) that cancut the DNA into fragments. Restriction enzymes are sequence specific: they recognize and bindonly to specific DNA sequences and cut the sugar-phosphate backbones of the DNA strands.For example, EcoRI recognizes and binds only to the sequence GAATTC. A different enzyme,called DNA ligase, can attach or rejoin DNA fragments with complementary ends. It does thatby catalyzing the chemical reaction that rejoins the DNA sugar-phosphate bonds. This processis called ligation.2Figure 1: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96C. (2) Annealing at (eg)68C. (3) Elongation at 72C (P=Polymerase). (4) The first cycle is complete. The two resultingDNA strands make up the template DNA for the next cycle, thus doubling the amount of DNAduplicated for each new cycle (a total of three cycles is shown above). Image is taken fromWikipedia.References:• http://www.dnalc.org/ddnalc/resources/restriction.html4 Electroporation and Transformation3DNA transformation is a naturally occurring event in which DNA can be transferred intobacteria. This event, however, is very rare and, for this reason, a method has been develop ed inorder to make bacterial cells more ”competent” for transforming foreign DNA.The cell membranes of bacteria have hundreds of p ores (i.e. adhesion zones) but themembranes themselves are made of lipid molecules that have negatively charged phosphates.So, even though the pores are large enough to admit small loops of DNA, the negatively chargedDNA is repelled by the lipids of the cell membrane. In order to shield the negative charges,calcium is added (Ca2+). Calcium interacts with the lipids’ and the DNA’s negative charge,creating a neutral situation. The lowering of the temperature congeals the membrane and makesit easier to shield the negative charges of the lipids. Then, a ”heat shock”, (i.e. a quick risein temperature), creates a temperature imbalance on either side of the bacterial membrane andsets up a current. The DNA can then be swept through the adhesion zone, since there are noelectrostatic interactions to prevent that any more. The transfer of the DNA into the bacterialcell has successfully taken place.Electroporation is also used instead of a heat sho ck. When the voltage across the mem-brane exceeds its dielectric strength, then pores are formed. If the electric field and/or theduration of exposure to this field are really short, then the pores can reseal after a short periodof time. However, this p eriod of time is long enough for the DNA to enter into the cell and,therefore, for the transformation to take place!References:• http://en.wikipedia.org/wiki/Electroporation•


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